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Methodology
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2
All foods were prescreened using MFHPB–30 to ensure that no naturally contaminating
L. monocytogenes
was present. An aerobic
3
plate count was conducted to evaluate the level of background flora present in each product (Table 10).
4
5
Strains of
L. monocytogenes
,
L. innocua
, and
E. faecalis
were cultured by inoculating BHI and incubating at 37 ± 2°C for 24 hours.
6
Following incubation, the strains to be used for inoculating the food matrices were heat-stressed by incubating at 55°C for 10 minutes
7
in a water bath to achieve 50 – 80% injury. The degree of injury of the culture was estimated by plating an aliquot of diluted culture
8
onto OXA and TSA agars. The agars were incubated at 37°C for 24 h and the colonies were counted. The degree of injury was
9
estimated as
100 )
1(
x
n
n
nonselect
select
−
, where
n
select
= number of colony forming units (cfu) on selective agar and
n
nonselect
= number of cfu on
10
nonselective agar. The resulting estimated injury for each strain can be found in Table 2.
11
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Each bulk food matrix was divided into three subsamples; 1) one subsample was set aside as an uninoculated control; 2) one
13
subsample was inoculated with the heat-stressed
L. monocytogenes
culture at a target concentration of 0.2 – 2 cfu/25 g; and 3) one
14
subsample was inoculated with the heat-stressed
L. monocytogenes
culture at a target concentration of 2 – 5 cfu/25 g. The first set of
15
hot dogs (Hot Dogs – 1) were inoculated with
L. innocua
as the competitor and the second set (Hot Dogs – 2) were inoculated with
16
Enterococcus faecalis
such that each competitor was at 10 times the concentration of
L. monocytogenes
. Following inoculation, the
17
subsamples were stabilized at 4°C for approximately 48 h during shipping to ensure that all samples were received by Monday of the
18
testing week. Thirty-g test portions were packaged from each subsample. Each sample set consisted of 5 replicate test portions of
19
uninoculated material, 20 replicate test portions of low level inoculated material, and 20 replicate test portions of high level inoculated
20
material. The test portions were randomized, blind-coded and shipped refrigerated to participants via overnight courier. All test
21
portions were packaged and shipped according to IATA Category B Biological Substances guidelines.
22
Upon receipt, the temperature of the samples were checked and recorded on the Sample Receipt Submission form provided and sent
23
to the independent laboratory for verification. Sample sets were stored at 2 – 8°C upon arrival until the time of analysis. Analyses
24
began approximately 72 h after inoculation of the matrix. The VIDAS LMX method and the reference method were carried out as
25
written. In the case of unpaired test portions, all VIDAS LMX enrichments, regardless of presumptive results, were struck to
26
selective agar for confirmation of typical colonies as described in the MFHPB – 30 method.
27
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The most probable number (MPN) was estimated for each level by the independent laboratory and was calculated based on the
29
probability of detection (POD) of the reference method across all labs using the AOAC MPN calculator. Enrichments, isolation and
30
confirmations were carried out according to the MFHPB – 30 reference method.
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PTM Certification Report