414 / 596
hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAA and
1
incubated at 37 ± 1°C for 24 ± 3 hours. PALCAM and OAA agar plates were examined for the
2
presence of suspect colonies. If no suspect colonies were present, the agar plate was re-incubated
3
for an additional 18-24 hours at 37 ± 1°C. If no suspect colonies present the sample was
4
determined to be negative for
Listeria.
If suspect colonies were present on the PALCAM or
5
OAA agar plates, these suspect colonies were streaked to HBO and incubated at 37 ± 1
o
C for 18-
6
24 hours. HBO plates were examined for hemolysis reactions and well-isolated colonies were
7
transferred to BHI broth and incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth
8
were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and
9
examined for morphology by preparing a Gram stain. Additionally, purified HBO isolates were
10
identified using the VITEK
®
GP Biochemical Identification following AOAC OMA 2013.02.
11
12
13
3M
™
Molecular Detection Assay 2 - Listeria
14
15
All 25 g samples were analyzed by the 3M
™
MDA 2 -
Listeria
were enriched with 225 mL of
16
Demi-Fraser Broth with the addition of ferric ammonium citrate; all test portions were then
17
homogenized by stomaching thoroughly for 2 ± 0.2 minutes and incubated at 37 ± 1 °C for 24-28
18
hours. For the 125 g deli turkey test portions, samples were enriched with 1125 mL of Demi-
19
Fraser Broth with the addition of ferric ammonium citrate; test portions were then homogenized
20
by stomaching thoroughly for 2 ± 0.2 minutes and incubated at 37 ± 1 °C for 24 hours. For
21
whole melons, test portions were enriched with approximately 1.5 times the weight of the whole
22
melon in Demi Fraser with ferric ammonium citrate and incubated at 37 ± 1 °C for 26 hours.
23
Stainless steel sponge samples were enriched with 225 mL of Demi-Fraser Broth with the
24
addition of ferric ammonium citrate; samples were homogenized by hand thoroughly for 2 ± 0.2
25
minutes and incubated at 37 ±1 °C for 24 and26 hours. Sealed concrete environmental surface
26
sponge samples were enriched with 100 mL of Demi-Fraser Broth with the addition of ferric
27
ammonium citrate; samples were thoroughly homogenized by hand for 2 ± 0.2 minutes and
28
incubated at 37°C for 24 and 26 hours. Plastic environmental surface swab samples were
29
enriched with 10 mL of Demi-Fraser Broth with the addition of ferric ammonium citrate;
30
samples were homogenized by vortexing for 2 ± 0.2 minutes and incubated at 37 ±1 °C for 24
31
and 26 hours.
32
33
Prior to analysis, lysis tubes were brought to room temperature (20-25
o
C) by placing the tubes on
34
a sanitized bench top for 16-18 hours. Lysis tubes were inverted to mix up to four hours before
35
use. The lysis tubes were then de-capped and the rubber cap was discarded. A 20 µL aliquot of
36
each sample was transferred to separate lysis tubes; a new pipette tip was used after each sample
37
transfer. Uncovered samples were placed in the 3M Molecular Detection Heat Block Insert and
38
heated for15±1 minutes at 100 ± 1
o
C. Following the heat lysis, samples were transferred into the
39
Chill Block Insert and placed on a sanitized laboratory bench and were allowed to cool at 18-
40
28°C for 5-10 minutes.
41
42
A 20 µL aliquot of each lysed sample and control was added to separate reagent tubes, and
43
samples were mixed by pipetting up and down five times. A Matrix Control tube was analyzed
44
with the samples for each matrix to verify that no interference with the assay was caused by the
45
matrix. Also lysed was a sterile Demi Fraser Broth with FAC tube for the kit Negative Control
46