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1
Internal Study
2
3
Inclusivity and Exclusivity
4
5
Methodology
6
Fiftyfrozen
Listeria monocytogenes
strainsuspensions were thawed and sub-cultured in brain heart
7
infusion (BHI) broth overnight at 37°C ± 1°C. The cultures were diluted in peptonesalt
8
solutionin order to inoculate between 10 to 100 cells per 225 mL of Demi Fraser. The
9
enrichment broths were incubated for 24 hours at 37°C ± 1°C, and the3M MDA2 -
10
Listeriamonocytogenes
method wasthen performed.
11
Thirty frozen non-
Listeriamonocytogenes
strain suspensions were thawed and sub-cultured
12
grown
in BHI broth overnight at 37°C ± 1°C. The cultures were diluted in buffered peptone water
13
(BPW) or de Man, Rogosa, and Sharpe (MRS) in order to inoculate 10
5
cells/mL. The broths
14
were then incubated for 24 hours at the appropriate incubation temperature in order to have
15
culture to test with the 3M MDA2 –
Listeria monocytogenes
method
(Table 3a)
.
16
17
An additional thirteen frozen non-
Listeria monocytogenes
strain suspensions were thawed, or
18
lyophilized cultures were revived, and then streaked to sheep blood agar (SBA). Single isolated
19
colonies were picked and sub-cultured in 10 mL Demi-Fraser broth with FAC for 24 hours at
20
37°C ± 1°C. The cultures were diluted with fresh Demi Fraser broth with FAC before testing
21
using the 3M MDA2 -
Listeria monocytogenes
kit (Table 3b).
22
23
Results
24
All 50
Listeria monocytogenes
strains were detected by the 3M MDA2 -
25
Listeriamonocytogenes
method. None of the
30 43
non-
Listeria monocytogenes
strains were
26
detected. See Tables 2 and 3
a, 3b
for study details and results.
27
28
29
Matrix Study
30
31
The matrix study consisted of evaluating a total of 30 un-paired sample replicates for 11
32
matrices, along with a raw chicken leg pieces evaluating 20 sample replicates using two separate
33
lots. Within each sample set, there were 5 un
-
inoculated samples (0 CFU/test portion), 20 low
34
level inoculated samples (0.2-2 CFU/test portion), and 5 high level inoculated samples (2-5
35
CFU/test portion), except for raw chicken leg pieces that were naturally contaminated with the
36
target analyte. The inoculum was prepared by transferring a single
Listeriamonocytogenes
colony
37
from Trypticase soy agar with 5% sheep blood (SBA) into BHI broth and incubating the culture
38
at 35 ± 2
o
C for 24 ± 2 hours. Table 4 presents the sample preparation guidelines for the matrix.
39
All matrices were screened for the presence of the target organism following the appropriate
40
reference method. Additionally, an aerobic plate count (APC) was conducted following the
41
FDA/BAM Chapter 3 reference [9] method to determine the level of background flora in each
42
test matrix prior to inoculation.
43
Prior to inoculation of beef hot dogs, deli turkey, and queso fresco the broth culture inoculum
44
was heat stressed for 10 ± 1 minute at 50 ± 1°C in a water bath. The degree of injury of the
45
culture was estimated by plating an aliquot of diluted culture onto modified Oxford agar (MOX)
46
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