167 / 596
was inverted so that the inoculated end was on the bottom of the sterile bag. The bag was tied
1
closed and held for 48-72 hours at 2-8 °C.
2
3
For environmental surfaces, inocula were prepared by transferring a pure isolated colony of the
4
specified organism from SBA into BHI broth and incubated at 35 ± 2
o
C for 24 ± 2 hours.
5
Following incubation, serial dilutions were performed in BHI broth to achieve the target level
6
inoculum.
7
8
For stainless steel and sealed concrete surfaces, 4” x 4” areas were inoculated with 0.25 mL of
9
diluted
Listeria monocytogenes
culture. Stainless steel was also inoculated with competitor
10
organism
Enterococcus faecium
ATCC 19434 at 10x the level of the target organism. For plastic
11
surfaces, 1” x 1” areas were inoculated with 0.10 mL of diluted
Listeria seeligeri
culture. Plastic
12
was also inoculated with a competitor organism,
Enterococcus faecalis
ATCC 29212, at 10x the
13
level of the target organism. For the uninoculated test portions, sterile BHI broth was applied to
14
the test area. Each surface was allowed to dry for 16-24 hours at room temperature (24 ±2
o
C).
15
16
The 3M™ Hydrated Sponge Stick (pre-moistened with Dey-Engley) (stainless steel and sealed
17
concrete) and 3M Tecra Enviroswabs (pre-wetted with Letheen) (plastic) were sampled by using
18
horizontal and vertical sweeping motions. The sponges and the swabs were held at room
19
temperature for 2 hours prior to analysis. To determine the inoculation level for the
20
environmental surfaces, aliquots of each inoculum were plated on TSA in triplicate.
21
22
The level of
Listeria monocytogenes
in the low level inoculum was determined by Most
23
Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x 25 g (reference
24
method test portions), and 5 x 10 g inoculated test samples. For the high inoculation level, the
25
MPN was determined by examining 5 x 50 g, 5 x 25 g (reference method test portions) and 5 x
26
10 g . The level of
Listeria
in the low level inoculum for all 125 g test portions was determined
27
by MPN by evaluating 5 x 250 g, 20 or 5 x 125 g (reference method test portions), and 5 x 50 g
28
inoculated test samples. For the high inoculation level, the MPN was determined by examining 5
29
x 250 g, 5 x 125 g (reference method test portions), and 5 x 50 g inoculated test samples. Each
30
test portion was enriched with the reference method enrichment broth at the reference method
31
dilution scheme and analyzed by the reference method procedure. See Table A for details. The
32
number of positives from the 3 test levels was used to calculate the MPN using the LCF MPN
33
calculator (version 1.6) provided by AOAC RI. [11]
34
(http://www.lcfltd.com/customer/LCFMPNCalculator.exe)35
36
Table A: MPN Test Portion Sizes
37
Reference
Method Test
Portion
Inoculation
Level
MPN Test Portions
25 g
Low
5 x 50 g
20 x 25 g*
5 x 10 g
High
5 x 50 g
5 x 25 g*
5 x 10 g
125 g
Low
5 x 250 g
20 x 125 g*
5 x 50 g
High
5 x 250 g
5 x 125 g*
5 x 50 g
*
Test portions from reference method
38
39
40
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only