544 / 596
and Tryptic Soy agar (TSA). The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the
1
colonies were counted. The degree of injury was estimated as:
2
3
4
5
Where
n
select
= number of colonies on selective agar and
n
nonselect
= number of colonies on non-
6
selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to
7
yield fractional positive results (5-15 positive results) and a high level expected to yield all
8
positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and
9
held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the
10
organism to equilibrate within the sample.
11
For melons, a single whole melon was placed into a large sterile bag and the blossom end of
12
the melon was inoculated with 100 µL of the diluted
Listeria monocytogenes
culture. The liquid
13
culture was then allowed to soak into the melon. The melon was then inverted so that the
14
inoculated end was on the bottom of the sterile bag. The bag was tied closed and held for 48-72
15
hours at 2-8 °C.
16
For environmental surfaces, inocula were prepared by transferring a pure isolated colony of
17
the specified organism from SBA into BHI broth and incubated at 35± 2
o
C for 24 ± 2 hours.
18
Following incubation, serial dilutions were performed in BHI broth to achieve the target level
19
inoculum.
20
For stainless steel and sealed concrete surfaces, 4” x 4” areas were inoculated with 0.25 mL
21
of diluted
Listeria monocytogenes
culture. Stainless steel was also inoculated with competitor
22
organism
Enterococcus faecium
ATCC 19434 at 10x the level of the target organism. For plastic
23
surfaces, 1” x 1” areas were inoculated with 0.10 mL of diluted
Listeria monocytogenes
culture.
24
Plastic was also inoculated with a competitor organism,
Enterococcus faecalis
ATCC 29212, at
25
10x the level of the target organism. For the un
-
inoculated test portions, sterile BHI broth was
26
applied to the test area. Each surface was allowed to dry for 16-24 hours at room temperature (24
27
±2
o
C).
28
The 3M hydrated sampling sponges (pre-moistened with Dey-Engley) (stainless steel and
29
sealed concrete) and 3M™ Tecra™ Enviro Swabs (pre-wetted with Letheen) (plastic) were
30
sampled by using horizontal and vertical sweeping motions. The sponges and the swabs were
31
held at room temperature for 2 hours prior to analysis. To determine the inoculation level for the
32
environmental surfaces, aliquots of each inoculum were plated on TSA in triplicate.
33
The level of
Listeria monocytogenes
in the low level inoculum and high level inoculum was
34
determined by Most Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x
35
25 g (reference method test portions), and 5 x 10 g inoculated test samples for the low
36
inoculation level and by examining 5 x 25 g, 5 x 5 g and 5 x 1 g for the high inoculation level.
37
The level of
Listeria monocytogenes
in the low level inoculum and high level inoculum for all
38
125 g test portions was determined by MPN by evaluating 5 x 250 g, 20 or 5 x 125 g (reference
39
method test portions), and 5 x 50 g inoculated test samples. Each test portion was enriched with
40
the reference method enrichment broth at the reference method dilution scheme and analyzed by
41
the reference method procedure. The number of positives from the 3 test levels was used to
42
calculate the MPN using the LCF MPN calculator (version 1.6) provided by AOAC RI. [10]
43
(http://www.lcfltd.com/customer/LCFMPNCalculator.exe)44
45
100 )
1(
x
n
n
nonselect
select
−