166 / 596
Results
1
2
All 55
Listeria
strains were detected by the 3M MDA 2 -
Listeria
method.
L. grayi
recovery was
3
enhanced when a food sample (Ultra-high-temperature [UHT] milk as described in ISO 16140
4
[9]) was added to the medium in the standard 1:10 dilution scheme. None of the 30 non-
Listeria
5
strains were detected. See Tables 2 and 3 for study details and results.
6
7
8
Matrix Study
9
10
The method comparison study consisted of evaluating a total of 30 un-paired sample replicates
11
for 10 matrixes, along with naturally contaminated raw chicken (leg pieces), tested at the
12
independent laboratory, evaluating 20 sample replicates using two separate lots, see Table 4a.
13
The raw chicken (leg pieces and fillets) analyzed at the internal lab was inoculated according to
14
AOAC guidelines which are described below, see Table 4b. Within each sample set, there were
15
5 uninoculated samples (0 CFU/test portion), 20 low level inoculated samples (0.2-2 CFU/test
16
portion), and 5 high level inoculated samples (2-5 CFU/test portion), except for the naturally
17
contaminated raw chicken (leg pieces). The inoculum was prepared by transferring a single
18
Listeria
colony from Trypticase Soy Agar with 5% Sheep Blood (SBA) into BHI broth and
19
incubating the culture at 35 ± 2
o
C for 24 ± 2 hours. Tables 4a and b presents the sample
20
preparation guidelines for the matrix.
21
22
All matrixes were screened for the presence of the target organism following the appropriate
23
reference method. Additionally, an aerobic plate count (APC) was conducted following the
24
FDA/BAM Chapter 3 reference [10] method to determine the level of background flora in each
25
test matrix prior to inoculation.
26
27
Prior to inoculation of beef hot dogs, deli turkey, and queso fresco the broth culture inoculum
28
was heat stressed for 10 ± 1 minute at 50 ± 1°C in a water bath. The degree of injury of the
29
culture was estimated by plating an aliquot of diluted culture onto Modified Oxford Agar (MOX)
30
and Tryptic Soy Agar (TSA). The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the
31
colonies were counted. The degree of injury was estimated as:
32
33
100 )
1(
x
n
n
nonselect
select
−
34
35
Where
n
select
= number of colonies on selective agar and
n
nonselect
= number of colonies on non-
36
selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to
37
yield fractional positive results (5-15 positive results) and a high level expected to yield all
38
positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and
39
held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the
40
organism to equilibrate within the sample.
41
42
For the inoculation of the whole melons, a single whole melon was placed into a large sterile bag
43
and the blossom end of the melon was inoculated with 100 µL of the diluted
Listeria
44
monocytogenes
culture. The liquid culture was then allowed to soak into the melon. The melon
45
AOAC Research Institute
Expert Review Panel U e Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only