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plates were examined for hemolysis. Final confirmation was conducted using the VITEK
®
GP
1
Biochemical Identification card following AOAC OMA 2013.02.
2
3
AOAC 993.12 Listeria Reference Method
4
5
Twenty-five gram test portions were enriched in 225 mL ± 5 mL of selective enrichment
6
medium, containing yeast extract (1.35 g/225 mL), acriflavine mono hydrochloride (1.125 g/225
7
mL), nalidixic acid (sodium salt) solution (1.125 g/225 mL), and cycloheximide (2.25 g/225
8
mL), and incubated at 30 ± 1 °C for 48 hours. The enriched samples were then streaked to
9
Oxford Agar (OXA) and incubated at 37 ± 1°C for 48 hours. At 48 hours, the OXA plates were
10
examined for suspect colonies. If present, up to 5 suspect colonies were streaked to TSA/YE to
11
obtain well isolated, pure colonies. Pure colonies on TSA/YE were analyzed for Gram-stain
12
reaction and for catalase activity. Catalase positive, Gram positive rods colonies were then
13
stabbed to SBA and incubated at 37 ±1 °C for 48 hours. After 48 hours, the SBA plates were
14
examined for typical hemolytic reactions. The same colony picked to SBA was also transferred
15
to TSB/YE. The TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was
16
turbid, indicating sufficient growth. Another TSB/YE tube was inoculated with the same colony
17
and incubated at 37 ±1 °C for 24 hours to be used for carbohydrate utilization testing. The
18
TSB/YE tube incubated at 25 ±1 °C were used to prepare a wet mount slide to determine motility
19
pattern. From the TSB/YE tube incubated at 37 ± 1 °C, Motility Test Medium (MTM) was
20
stabbed and incubated at 25 ±1
o
C. After 2 days, and up to 7 days, the MTM tubes were observed
21
for umbrella-like growth. Additionally from the TSB/YE tube, a loopful (0.1 mL) was
22
transferred into carbohydrate fermentation broth (purple broth) containing 1.0 mL of either 5%
23
rhamnose or 5% xylose. The purple broth tubes were incubated at 37 ± 1 °C and examined for up
24
to 7 days.
25
26
ISO 11290-1/A1 Reference Method
27
28
For the ISO 11290-1/A1 reference method, 25 g test portions were enriched with 225 half Fraser
29
broth. All test portions were mechanically stomached for two minutes. The test portions were
30
incubated at 30 ± 1°C for 24 ± 3 hours. After incubation, 0.1 mL of the sample enrichment was
31
transferred to 10 mLFB containing 0.1 mL of 5% ferric ammonium citrate and incubated at 37 ±
32
1°C for 48 ± 3 hours. After 48 ± 3 hours, a loopful of the sample secondary FB enrichment was
33
streaked to PALCAM and OAA
(Ottovani-Agosti Agar
)and incubated at 37 ± 1°C for 24 ± 3
34
hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAAand
35
incubated at 37 ± 1°C for 24 ± 3 hours. PALCAM and OAA agar plates were examined for the
36
presence of suspect colonies. If no suspect colonies were present, the agar plate was re-incubated
37
for an additional 18-24 hours at 37 ± 1°C. If no suspect colonies present the sample was
38
determined to be negative for
Listeria.
If suspect colonies were present on the PALCAM or
39
OAA agar plates, these suspect colonies were streaked to HBO and incubated at 37 ± 1
o
C for 18-
40
24 hours. HBO plates were examined for hemolysis reactions and well-isolated colonies were
41
transferred to BHI broth and incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth
42
were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and
43
examined for morphology by preparing a Gram stain. Additionally, purified HBO isolates were
44
identified using the VITEK
®
GP Biochemical Identification following AOAC OMA 2013.02.
45
46
AOAC Research Institute
Expert Review Pane Use Only
OMAMAN-30 D/ PTM Validation Report 081501
OMA ERP - June 2016
ERP Use Only