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10ml Fraser both (FB) and incubated at 35±1°C for 48 h. Any FB showing blackening
1
(24-48h) was streaked to MOX agar. In addition, approximately 0.1ml of the UVM broth
2
(20-24h) was streaked directly to Modified Oxford agar (MOX) and ALOA agar then
3
incubated for 24-48h at 35±1°C. At least one typical colony per sample/media was
4
streaked to Horse blood overlay agar (HLA) and confirmed using standard biochemical
5
tests (FDA BAM).
6
FDA-BAM Chapter 10
7
Twenty 25g inoculated samples and five 25g uninoculated samples were added to 225 ml
8
of Buffered Listeria Enrichment broth base (BLEB). These were stomached for 2
9
minutes then incubated at 30±1°C. For cantaloupe melons, approximately 1 L of BLEB
10
was added to each melon and samples were allowed to soak for 1 h at room temperature.
11
For all matrices, after 4 h incubation the selective agents were added and incubation was
12
continued for another 44h at 30±1°C. At 24 and 48h selective enrichment broths were
13
streaked to Oxford agar plates, which were incubated for 24-48h at 35±1°C. Typical
14
colonies were confirmed (minimum1/sample).
15
AOAC 993.12
16
Twenty 25 gram portions of the cheese and ice cream were enriched with 225 ml of
17
selective enrichment medium (prepared per method instructions), mixed at high speed for
18
2 minutes and incubated at 30 ± 1°C for 48 hours. After 48 hours, the selective
19
enrichment was streaked to Oxford agar and incubated 37 ± 1°C for 48 hours.
20
Confirmation was according to AOAC 993.12.
21
Method Comparison Study – Environmental Surfaces
22
Four environmental surfaces were evaluated in this study. The VIDAS LPT was
23
compared to the USDA FSIS reference method. Since the USDA method and VIDAS
24
LPT method utilize different enrichment/repair broths, separate surface areas were
25
inoculated and sampled. There were 20 replicates for the inoculated level and 5 replicates
26
for the uninoculated level for each method. Surfaces were inoculated with the target
27
organism at a level to produce fractionally positive results for at least one of the methods.
28
The surface area sampled by sponges was 4” x 4” area or 100 cm
2
or by swabs was 1” x
29
1” or 5 cm
2
. Prior to inoculation, surfaces were decontaminated using 70% ethanol.
30
Cultures were inoculated into tryptone soy broth (TSB) and incubated at 30
°
C for 24 h
31
then diluted and spread plate counts were performed on trypticase soy agar (TSA).
32
Surfaces were inoculated (at a level expected to yield fractionally positive results) over
33
the entire test surface area, avoiding excessive accumulation of liquid that would dry
34
unevenly. In addition to the target organism, the four surfaces were inoculated with a
35
competitor,
Enterococcus faecalis
SM244 at a target: competitor ratio of 1:10.The surface
36
was allowed to dry for 16-24 h at room temperature.
37
38
49