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TSA/YE plates were incubated at 35 ± 1°C for 24-48 hours and then examined for purity. Pure
1
colonies were tested for catalase reactivity and a Gram Stain was conducted. A pure
Listeria
2
colony was transferred to Trypticase Soy Broth containing 0.6% yeast extract (TSB/YE). The
3
TSB/YE cultures were incubated at 25 ± 1°C overnight, or until the broth was turbid, indicating
4
sufficient growth. Catalase-positive organisms were stabbed into plates of 5% Sheep Blood Agar
5
(SBA) and incubated at 35 ± 1°C for 24-48 hours. The TSB/YE tubes incubated at 25 ±1°C were
6
used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA plates
7
were examined for hemolysis. Final confirmation was conducted using the VITEK
®
GP
8
Biochemical Identification card following AOAC OMA 2013.02.
9
10
AOAC 993.12 Listeria Reference Method
11
12
Twenty-five gram test portions were enriched in 225 mL ± 5 mL of selective enrichment
13
medium, containing yeast extract (1.35 g/225 mL), acriflavine mono hydrochloride (1.125 g/225
14
mL), nalidixic acid (sodium salt) solution (1.125 g/225 mL), and cycloheximide (2.25 g/225
15
mL), and incubated at 30 ± 1 °C for 48 hours. The enriched samples were then streaked to
16
Oxford Agar (OXA) and incubated at 37 ± 1°C for 48 hours. At 48 hours, the OXA plates were
17
examined for suspect colonies. If present, up to 5 suspect colonies were streaked to TSA/YE to
18
obtain well isolated, pure colonies. Pure colonies on TSA/YE were analyzed for Gram-stain
19
reaction and for catalase activity. Catalase positive, Gram positive rods colonies were then
20
stabbed to SBA and incubated at 37 ±1 °C for 48 hours. After 48 hours, the SBA plates were
21
examined for typical hemolytic reactions. The same colony picked to SBA was also transferred
22
to TSB/YE. The TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was
23
turbid, indicating sufficient growth. Another TSB/YE tube was inoculated with the same colony
24
and incubated at 37 ±1 °C for 24 hours to be used for carbohydrate utilization testing. The
25
TSB/YE tube incubated at 25 ±1 °C was used to prepare a wet mount slide to determine motility
26
pattern. From the TSB/YE tube incubated at 37 ± 1 °C, Motility Test Medium (MTM) was
27
stabbed and incubated at 25 ±1
o
C. After 2 days, and up to 7 days, the MTM tubes were observed
28
for umbrella-like growth. Additionally from the TSB/YE tube, a loopful (0.1 mL) was
29
transferred into carbohydrate fermentation broth (purple broth) containing 1.0 mL of either 5%
30
rhamnose or 5% xylose. The purple broth tubes were incubated at 37 ± 1 °C and examined for up
31
to 7 days.
32
33
ISO 11290-1/A1 Reference Method
34
35
For the ISO 11290-1/A1 reference method, 25 g test portions were enriched with 225 half Fraser
36
broth. All test portions were mechanically stomached for two minutes. The test portions were
37
incubated at 30 ± 1°C for 24 ± 3 hours. After incubation, 0.1 mL of the sample enrichment was
38
transferred to 10 mL FB containing 0.1 mL of 5% ferric ammonium citrate and incubated at 37 ±
39
1°C for 48 ± 3 hours. After 48 ± 3 hours, a loopful of the sample secondary FB enrichment was
40
streaked to PALCAM and Ottovani-Agosti Agar (OAA) and incubated at 37 ± 1°C for 24 ± 3
41
hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAA and
42
incubated at 37 ± 1°C for 24 ± 3 hours. PALCAM and OAA agar plates were examined for the
43
presence of suspect colonies. If no suspect colonies were present, the agar plate was re-incubated
44
for an additional 18-24 hours at 37 ± 1°C. If no suspect colonies present the sample was
45
determined to be negative for
Listeria.
If suspect colonies were present on the PALCAM or
46
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only