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3M MDA 2
Listeria
species and
L. monocytogenes
Collaborative Study
OMA-2016-MONTH-XXX
5/4/16
2.14.3
Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of
1
the enriched sample over the surface of the plate. Incubate the MOX at 35
2
± 2°C for 26 ±2 h.
3
2.14.4
Examine the MOX plates for colonies with morphology typical of
Listeria
4
spp
.
At 26 ± 2 h, suspect colonies are typically small (ca. 1 mm) and are
5
surrounded by a zone of darkening due to esculin hydrolysis.
6
2.14.5
If suspect colonies are present on MOX, transfer suspect colonies to HL
7
agar.
8
2.14.6
If no suspect colonies are evident, re-incubate the MOX plate for an
9
additional 26 ± 2 hour.
10
2.14.7
After 26 ± 2 h of incubation, examine the FB for the potential presence of
11
Listeria spp.,
by visual examination of the broth for darkening due to
12
esculin hydrolysis.
13
2.14.8
If any degree of FB darkening is evident, aseptically dispense a drop
14
approximating 0.1 ± 0.02 ml of FB onto a MOX plate. Swab or streak 25-
15
40% of the surface of the MOX plate with the FB inoculum. Use a loop to
16
streak for isolation from the initial swab/streak quadrant onto the
17
remainder of the plate. Incubate the MOX plate at 35 ± 2°C for 26 ± 2 h.
18
2.14.9
If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a total
19
incubation time of 48 ± 2 h has been achieved.
20
2.14.10Re-examine the FB for evidence of darkening after 48 ± 2 h of total
21
incubation. If any degree of darkening is evident, swab, streak and
22
incubate a MOX plate.
23
2.14.11If no darkening of FB is evident and no suspect MOX and/or HL colonies
24
have been demonstrated, the sample is considered negative for
Listeria
25
spp.
26
2.14.12If suspect colonies are present on MOX from any source, streak for
27
isolation on one or more HL agar plates. Incubate the streaked HL at 35 ±
28
2°C for 22 ± 4 h.
29
2.14.13After incubation, examine the HL plate(s) against backlight for translucent
30
colonies surrounded by a small zone of β-hemolysis.
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2.14.14If at least one suspect colony is clearly isolated, proceed to confirmatory
32
testing. Hold all HL plates containing suspect colonies (room temperature
33
or refrigeration) until confirmatory testing is complete.
34
2.14.15If suspect colonies or β-hemolytic growth are present on HL but not
35
clearly isolated, re-streak representative suspect colonies/growth onto one
36
or more fresh HL plates and incubate at 35 ± 2°C for 22 ± 4 h.
37
2.14.16Confirm ALL test portions according to the USDA FSIS MLG 8.09
38
biochemical/serological procedures. Biochemical tests will be performed
39
using API or VITEK 2 GP. Please follow manufacturer’s instructions
40
when using any of these rapid methods.
41
42
USDA FSIS Reference Method
43
1.17
Add 25g test portion to 225 mL UVM broth. Stomach (Seward 400 or equivalent)
44
for 2± 0.2 minutes and incubate for 20-26h at 30
±
2
°
C.
45
AOAC Research Institute
Exp rt Review Panel Use Only
OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol
OMA ERP - June 2016
ERP Use Only