410 / 596
Thirty frozen non-
Listeria
strain suspensions were thawed and sub-cultured in BHI broth
1
overnight at 37°C ± 1°C. The cultures were diluted in Buffered Peptone Water (BPW) or de
2
Man, Rogosa, and Sharpe (MRS) in order to inoculate 10
5
cells/mL. The broths were then
3
incubated for 24 hours at the appropriate incubation temperature in order to have culture to test
4
with the 3M MDA 2 -
Listeria
method. See Table 3.
5
6
Results
7
8
All
55 63
Listeria
strains were detected by the 3M MDA 2 -
Listeria
method.
L. grayi
recovery
9
was enhanced when a food sample (Ultra-high-temperature [UHT] milk as described in ISO
10
16140 [9]) was added to the medium in the standard 1:10 dilution scheme. None of the 30 non-
11
Listeria
strains were detected. See Tables 2
a, 2b
and 3 for study details and results.
12
13
14
Matrix Study
15
16
The method comparison study consisted of evaluating a total of 30 un-paired sample replicates
17
for 10 matrixes, along with naturally contaminated raw chicken (leg pieces), tested at the
18
independent laboratory, evaluating 20 sample replicates using two separate lots, see Table 4a.
19
The raw chicken (leg pieces and fillets) analyzed at the internal lab was inoculated according to
20
AOAC guidelines which are described below, see Table 4b. Within each sample set, there were
21
5 uninoculated samples (0 CFU/test portion), 20 low level inoculated samples (0.2-2 CFU/test
22
portion), and 5 high level inoculated samples (2-5 CFU/test portion), except for the naturally
23
contaminated raw chicken (leg pieces). The inoculum was prepared by transferring a single
24
Listeria
colony from Trypticase Soy Agar with 5% Sheep Blood (SBA) into BHI broth and
25
incubating the culture at 35 ± 2
o
C for 24 ± 2 hours. Tables 4a and b presents the sample
26
preparation guidelines for the matrix.
27
28
All matrixes were screened for the presence of the target organism following the appropriate
29
reference method. Additionally, an aerobic plate count (APC) was conducted following the
30
FDA/BAM Chapter 3 reference [10] method to determine the level of background flora in each
31
test matrix prior to inoculation.
32
33
Prior to inoculation of beef hot dogs, deli turkey, and queso fresco the broth culture inoculum
34
was heat stressed for 10 ± 1 minute at 50 ± 1°C in a water bath. The degree of injury of the
35
culture was estimated by plating an aliquot of diluted culture onto Modified Oxford Agar (MOX)
36
and Tryptic Soy Agar (TSA). The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the
37
colonies were counted. The degree of injury was estimated as:
38
39
100 )
1(
x
n
n
nonselect
select
−
40
41
Where
n
select
= number of colonies on selective agar and
n
nonselect
= number of colonies on non-
42
selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to
43
yield fractional positive results (5-15 positive results) and a high level expected to yield all
44
positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and
45