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held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the
1
organism to equilibrate within the sample.
2
3
For the inoculation of the whole melons, a single whole melon was placed into a large sterile bag
4
and the blossom end of the melon was inoculated with 100 µL of the diluted
Listeria
5
monocytogenes
culture. The liquid culture was then allowed to soak into the melon. The melon
6
was inverted so that the inoculated end was on the bottom of the sterile bag. The bag was tied
7
closed and held for 48-72 hours at 2-8 °C.
8
9
For environmental surfaces, inocula were prepared by transferring a pure isolated colony of the
10
specified organism from SBA into BHI broth and incubated at 35 ± 2
o
C for 24 ± 2 hours.
11
Following incubation, serial dilutions were performed in BHI broth to achieve the target level
12
inoculum.
13
14
For stainless steel and sealed concrete surfaces, 4” x 4” areas were inoculated with 0.25 mL of
15
diluted
Listeria monocytogenes
culture. Stainless steel was also inoculated with competitor
16
organism
Enterococcus faecium
ATCC 19434 at 10x the level of the target organism. For plastic
17
surfaces, 1” x 1” areas were inoculated with 0.10 mL of diluted
Listeria seeligeri
culture. Plastic
18
was also inoculated with a competitor organism,
Enterococcus faecalis
ATCC 29212, at 10x the
19
level of the target organism. For the uninoculated test portions, sterile BHI broth was applied to
20
the test area. Each surface was allowed to dry for 16-24 hours at room temperature (24 ±2
o
C).
21
22
The 3M™ Hydrated Sponge Stick (pre-moistened with Dey-Engley) (stainless steel and sealed
23
concrete) and 3M Tecra Enviroswabs (pre-wetted with Letheen) (plastic) were sampled by using
24
horizontal and vertical sweeping motions. The sponges and the swabs were held at room
25
temperature for 2 hours prior to analysis. To determine the inoculation level for the
26
environmental surfaces, aliquots of each inoculum were plated on TSA in triplicate.
27
28
The level of
Listeria monocytogenes
in the low level inoculum was determined by Most
29
Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x 25 g (reference
30
method test portions), and 5 x 10 g inoculated test samples. For the high inoculation level, the
31
MPN was determined by examining 5 x 50 g, 5 x 25 g (reference method test portions) and 5 x
32
10 g . The level of
Listeria
in the low level inoculum for all 125 g test portions was determined
33
by MPN by evaluating 5 x 250 g, 20 or 5 x 125 g (reference method test portions), and 5 x 50 g
34
inoculated test samples. For the high inoculation level, the MPN was determined by examining 5
35
x 250 g, 5 x 125 g (reference method test portions), and 5 x 50 g inoculated test samples. Each
36
test portion was enriched with the reference method enrichment broth at the reference method
37
dilution scheme and analyzed by the reference method procedure. See Table A for details. The
38
number of positives from the 3 test levels was used to calculate the MPN using the LCF MPN
39
calculator (version 1.6) provided by AOAC RI. [11]
40
(http://www.lcfltd.com/customer/LCFMPNCalculator.exe)41
42
Table A: MPN Test Portion Sizes
43
Reference
Method Test
Portion
Inoculation
Level
MPN Test Portions
25 g
Low
5 x 50 g
20 x 25 g*
5 x 10 g