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3M MDA 2
Listeria
species and
L. monocytogenes
Collaborative Study
OMA-2016-MONTH-XXX
5/4/16
1.18
Transfer 0.1 ± 0.02 ml of the enriched sample to 10 ± 0.5 ml of Fraser Broth (FB).
1
As per media preparation instructions, be sure that appropriate supplements have
2
been added to the FB prior to inoculation. Incubate inoculated FB tubes at 35 ±
3
2°C for 26 ± 2 h.
4
1.19
Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of the
5
enriched sample over the surface of the plate. Incubate the MOX at 35 ± 2°C for
6
26 ±2 h.
7
1.20
Examine the MOX plates for colonies with morphology typical of
Listeria
spp
.
At
8
26 ± 2 h, suspect colonies are typically small (ca. 1 mm) and are surrounded by a
9
zone of darkening due to esculin hydrolysis.
10
1.21
If suspect colonies are present on MOX, transfer suspect colonies to HL agar.
11
1.22
If no suspect colonies are evident, re-incubate the MOX plate for an additional 26
12
± 2 hour.
13
1.23
After 26 ± 2 h of incubation, examine the FB for the potential presence of
Listeria
14
spp.,
by visual examination of the broth for darkening due to esculin hydrolysis.
15
1.24
If any degree of FB darkening is evident, aseptically dispense a drop
16
approximating 0.1 ± 0.02 ml of FB onto a MOX plate. Swab or streak 25-40% of
17
the surface of the MOX plate with the FB inoculum. Use a loop to streak for
18
isolation from the initial swab/streak quadrant onto the remainder of the plate.
19
Incubate the MOX plate at 35 ± 2°C for 26 ± 2 h.
20
1.25
If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a total
21
incubation time of 48 ± 2 h has been achieved.
22
1.26
Re-examine the FB for evidence of darkening after 48 ± 2 h of total incubation.
23
If any degree of darkening is evident, swab, streak and incubate a MOX plate.
24
1.27
If no darkening of FB is evident and no suspect MOX and/or HL colonies have
25
been demonstrated, the sample is considered negative for
Listeria
spp.
26
1.28
If suspect colonies are present on MOX from any source, streak for isolation on
27
one or more HL agar plates. Incubate the streaked HL at 35 ± 2°C for 22 ± 4 h.
28
1.29
After incubation, examine the HL plate(s) against backlight for translucent
29
colonies surrounded by a small zone of β-hemolysis.
30
1.30
If at least one suspect colony is clearly isolated, proceed to confirmatory testing.
31
Hold all HL plates containing suspect colonies (room temperature or
32
refrigeration) until confirmatory testing is complete.
33
1.31
If suspect colonies or β-hemolytic growth are present on HL but not clearly
34
isolated, re-streak representative suspect colonies/growth onto one or more fresh
35
HL plates and incubate at 35 ± 2°C for 22 ± 4 h.
36
1.32
Confirm ALL test portions according to the USDA FSIS biochemical/serological
37
procedures. Biochemical tests will be performed using API or VITEK 2 GP.
38
Please follow manufacturer’s instructions when using any of these rapid methods.
39
40
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Final Results
42
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All results are to be recorded on the data sheets provided. For each matrix test portion, record
44
the results reported by the 3M MDA 2 -
Listeria monocytogenes
and the 3M MDA 2-
Listeria
45
method along with the confirmation results for the 3M MDAs and reference methods.
46
AOAC Research Institute
Exp rt Review Panel Use Only
OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol
OMA ERP - June 2016
ERP Use Only