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colonies were tested for catalase reactivity and a Gram Stain was conducted. A pure
Listeria
1
colony was transferred to Trypticase Soy Broth containing 0.6% yeast extract (TSB/YE). The
2
TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was turbid, indicating
3
sufficient growth. Catalase-positive organisms were stabbed into plates of 5% Sheep Blood Agar
4
(SBA) and incubated at 35 ± 1°C for 24-48 hours. The TSB/YE tubes incubated at 25 ±1 °C
5
were used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA
6
plates were examined for hemolysis. Final confirmation was conducted using the VITEK
®
GP
7
Biochemical Identification card following AOAC OMA 2013.02.
8
9
AOAC 993.12 Listeria Reference Method
10
11
Twenty-five gram test portions were enriched in 225 mL ± 5 mL of selective enrichment
12
medium, containing yeast extract (1.35 g/225 mL), acriflavine mono hydrochloride (1.125 g/225
13
mL), nalidixic acid (sodium salt) solution (1.125 g/225 mL), and cycloheximide (2.25 g/225
14
mL), and incubated at 30 ± 1 °C for 48 hours. The enriched samples were then streaked to
15
Oxford Agar (OXA) and incubated at 37 ± 1°C for 48 hours. At 48 hours, the OXA plates were
16
examined for suspect colonies. If present, up to 5 suspect colonies were streaked to TSA/YE to
17
obtain well isolated, pure colonies. Pure colonies on TSA/YE were analyzed for Gram-stain
18
reaction and for catalase activity. Catalase positive, Gram positive rods colonies were then
19
stabbed to SBA and incubated at 37 ±1 °C for 48 hours. After 48 hours, the SBA plates were
20
examined for typical hemolytic reactions. The same colony picked to SBA was also transferred
21
to TSB/YE. The TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was
22
turbid, indicating sufficient growth. Another TSB/YE tube was inoculated with the same colony
23
and incubated at 37 ±1 °C for 24 hours to be used for carbohydrate utilization testing. The
24
TSB/YE tube incubated at 25 ±1 °C were used to prepare a wet mount slide to determine motility
25
pattern. From the TSB/YE tube incubated at 37 ± 1 °C, Motility Test Medium (MTM) was
26
stabbed and incubated at 25 ±1
o
C. After 2 days, and up to 7 days, the MTM tubes were observed
27
for umbrella-like growth. Additionally from the TSB/YE tube, a loopful (0.1 mL) was
28
transferred into carbohydrate fermentation broth (purple broth) containing 1.0 mL of either 5%
29
rhamnose or 5% xylose. The purple broth tubes were incubated at 37 ± 1 °C and examined for up
30
to 7 days.
31
32
ISO 11290-1/A1 Reference Method
33
34
For the ISO 11290-1/A1 reference method, 25 g test portions were enriched with 225 half Fraser
35
broth. All test portions were mechanically stomached for two minutes. The test portions were
36
incubated at 30 ± 1°C for 24 ± 3 hours. After incubation, 0.1 mL of the sample enrichment was
37
transferred to 10 mLFB containing 0.1 mL of 5% ferric ammonium citrate and incubated at 37 ±
38
1°C for 48 ± 3 hours. After 48 ± 3 hours, a loopful of the sample secondary FB enrichment was
39
streaked to PALCAM and OAA(Ottovani-Agosti Agar)and incubated at 37 ± 1°C for 24 ± 3
40
hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAAand
41
incubated at 37 ± 1°C for 24 ± 3 hours. PALCAM and OAA agar plates were examined for the
42
presence of suspect colonies. If no suspect colonies were present, the agar plate was re-incubated
43
for an additional 18-24 hours at 37 ± 1°C. If no suspect colonies present the sample was
44
determined to be negative for
Listeria.
If suspect colonies were present on the PALCAM or
45
OAA agar plates, these suspect colonies were streaked to HBO and incubated at 37 ± 1
o
C for 18-
46
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