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hours. After 24 hours of total incubation, the enriched samples were streaked to MOX agar plates
1
and incubated at 35 ±1 °C for 24-48 hours. The enriched samples were re-incubated for an
2
additional 24 hours at 30 ± 1 °C and then streaked to a second MOX agar plate which was
3
incubated for 24-48 hours at 35 ± 1
o
C. MOX agar plates were examined for suspect colonies, and
4
if present, at least 5 colonies were streaked to TSA containing 0.6% yeast extract (TSA/YE). The
5
TSA/YE plates were incubated at 35 ± 1°C for 24-48 hours and then examined for purity. Pure
6
colonies were tested for catalase reactivity and a Gram Stain was conducted. A pure
Listeria
7
colony was transferred to Trypticase Soy Broth containing 0.6% yeast extract (TSB/YE). The
8
TSB/YE cultures were incubated at 25 ± 1°C overnight, or until the broth was turbid, indicating
9
sufficient growth. Catalase-positive organisms were stabbed into plates of 5% Sheep Blood Agar
10
(SBA) and incubated at 35 ± 1°C for 24-48 hours. The TSB/YE tubes incubated at 25 ±1°C were
11
used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA plates
12
were examined for hemolysis. Final confirmation was conducted using the VITEK
®
GP
13
Biochemical Identification card following AOAC OMA 2013.02.
14
15
AOAC 993.12 Listeria Reference Method
16
17
Twenty-five gram test portions were enriched in 225 mL ± 5 mL of selective enrichment
18
medium, containing yeast extract (1.35 g/225 mL), acriflavine mono hydrochloride (1.125 g/225
19
mL), nalidixic acid (sodium salt) solution (1.125 g/225 mL), and cycloheximide (2.25 g/225
20
mL), and incubated at 30 ± 1 °C for 48 hours. The enriched samples were then streaked to
21
Oxford Agar (OXA) and incubated at 37 ± 1°C for 48 hours. At 48 hours, the OXA plates were
22
examined for suspect colonies. If present, up to 5 suspect colonies were streaked to TSA/YE to
23
obtain well isolated, pure colonies. Pure colonies on TSA/YE were analyzed for Gram-stain
24
reaction and for catalase activity. Catalase positive, Gram positive rods colonies were then
25
stabbed to SBA and incubated at 37 ±1 °C for 48 hours. After 48 hours, the SBA plates were
26
examined for typical hemolytic reactions. The same colony picked to SBA was also transferred
27
to TSB/YE. The TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was
28
turbid, indicating sufficient growth. Another TSB/YE tube was inoculated with the same colony
29
and incubated at 37 ±1 °C for 24 hours to be used for carbohydrate utilization testing. The
30
TSB/YE tube incubated at 25 ±1 °C was used to prepare a wet mount slide to determine motility
31
pattern. From the TSB/YE tube incubated at 37 ± 1 °C, Motility Test Medium (MTM) was
32
stabbed and incubated at 25 ±1
o
C. After 2 days, and up to 7 days, the MTM tubes were observed
33
for umbrella-like growth. Additionally from the TSB/YE tube, a loopful (0.1 mL) was
34
transferred into carbohydrate fermentation broth (purple broth) containing 1.0 mL of either 5%
35
rhamnose or 5% xylose. The purple broth tubes were incubated at 37 ± 1 °C and examined for up
36
to 7 days.
37
38
ISO 11290-1/A1 Reference Method
39
40
For the ISO 11290-1/A1 reference method, 25 g test portions were enriched with 225 half Fraser
41
broth. All test portions were mechanically stomached for two minutes. The test portions were
42
incubated at 30 ± 1°C for 24 ± 3 hours. After incubation, 0.1 mL of the sample enrichment was
43
transferred to 10 mL FB containing 0.1 mL of 5% ferric ammonium citrate and incubated at 37 ±
44
1°C for 48 ± 3 hours. After 48 ± 3 hours, a loopful of the sample secondary FB enrichment was
45
streaked to PALCAM and Ottovani-Agosti Agar (OAA) and incubated at 37 ± 1°C for 24 ± 3
46