284 / 596
Where
n
select
= number of colonies on selective agar and
n
nonselect
= number of colonies on non-
1
selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to
2
yield fractional positive results (5-15 positive results) and a high level expected to yield all
3
positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and
4
held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the
5
organism to equilibrate within the sample.
6
For melons, a single whole melon was placed into a large sterile bag and the blossom end of
7
the melon was inoculated with 100 µL of the diluted
Listeria monocytogenes
culture. The liquid
8
culture was then allowed to soak into the melon. The melon was then inverted so that the
9
inoculated end was on the bottom of the sterile bag. The bag was tied closed and held for 48-72
10
hours at 2-8 °C.
11
For environmental surfaces, inocula were prepared by transferring a pure isolated colony of
12
the specified organism from SBA into BHI broth and incubated at 35± 2
o
C for 24 ± 2 hours.
13
Following incubation, serial dilutions were performed in BHI broth to achieve the target level
14
inoculum.
15
For stainless steel and sealed concrete surfaces, 4” x 4” areas were inoculated with 0.25 mL
16
of diluted
Listeria monocytogenes
culture. Stainless steel was also inoculated with competitor
17
organism
Enterococcus faecium
ATCC 19434 at 10x the level of the target organism. For plastic
18
surfaces, 1” x 1” areas were inoculated with 0.10 mL of diluted
Listeria monocytogenes
culture.
19
Plastic was also inoculated with a competitor organism,
Enterococcus faecalis
ATCC 29212, at
20
10x the level of the target organism. For the uninoculated test portions, sterile BHI broth was
21
applied to the test area. Each surface was allowed to dry for 16-24 hours at room temperature (24
22
±2
o
C).
23
The 3M hydrated sampling sponges (pre-moistened with Dey-Engley) (stainless steel and
24
sealed concrete) and 3M™Tecra™ Enviro Swabs (pre-wetted with Letheen) (plastic) were
25
sampled by using horizontal and vertical sweeping motions. The sponges and the swabs were
26
held at room temperature for 2 hours prior to analysis. To determine the inoculation level for the
27
environmental surfaces, aliquots of each inoculum were plated on TSA in triplicate.
28
The level of
Listeria monocytogenes
in the low level inoculum and high level inoculum was
29
determined by Most Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x
30
25 g (reference method test portions), and 5 x 10 g inoculated test samples for the low
31
inoculation level and by examining 5 x 25 g, 5 x 5 g and 5 x 1 g for the high inoculation level.
32
The level of
Listeriamonocytogenes
in the low level inoculum and high level inoculum for all 125
33
g test portions was determined by MPN by evaluating 5 x 250 g, 20 or 5 x 125 g (reference
34
method test portions), and 5 x 50 g inoculated test samples. Each test portion was enriched with
35
the reference method enrichment broth at the reference method dilution scheme and analyzed by
36
the reference method procedure. The number of positives from the 3 test levels was used to
37
calculate the MPN using the LCF MPN calculator (version 1.6) provided by AOAC RI. [10]
38
(http://www.lcfltd.com/customer/LCFMPNCalculator.exe)39
40
41
USDA/FSIS MLG 8.09 Reference Method
42
43
For the USDA/FSIS MLG 8.09 reference method, 25 g test portions were enriched with 225 ± 5
44
mL of modified University of Vermont Medium broth (UVM) and 125 g test portions were
45
enriched with 1125 ± 25 mL of UVM. All test portions were mechanically stomached for two
46
minutes. The 25 g test portions were incubated at 30 ± 2 °C for 20-26 hours and the 125 g test
47
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-30 D/ PTM Validation Report 081501
OMA ERP - June 2016
ERP Use Only