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3M MDA 2
Listeria
species and
L. monocytogenes
Collaborative Study
OMA-2016-MONTH-XXX
5/4/16
1.6
Transfer 20 µL Negative Control [NC], sterile enrichment medium (e.g. Demi-
1
Fraser Broth) into a Lysis Solution [LS] tube after all enriched samples have been
2
completed. Do not use water as a NC.
3
1.7
Place uncovered LS tubes in 3M MDS Heat block, heat 15±1 min at 100±1°C.
4
LS Solution will change from pink (cool) to yellow (hot).
5
1.8
Place LS tubes (without rack lid) in chill block insert (at ambient temperature) for
6
5-10 min. Lysis solution in LS tube will revert to pink color.
7
1.9
Transfer 20 µL sample lysate from the upper portion of fluid in the LS tube into
8
regent tube. Mix gently by pipetting up and down 5 times.
9
1.10
Transfer 20 µL of NC lysate into a reagent tube.
10
1.11
Transfer 20 µL of NC lysate into a Reagent Control (RC) tube.
11
1.12
Load capped tubes into speed loader tray and close lid.
12
1.13
Start assay.
13
1.14
Validation Study Confirmation
14
1.14.1
Confirm each test portion, regardless of presumptive result.
15
1.14.2
Transfer 0.1 ± 0.02 ml of the enriched sample to 10 ± 0.5 ml of Fraser
16
Broth (FB). As per media preparation instructions, be sure that
17
appropriate supplements have been added to the FB prior to inoculation.
18
Incubate inoculated FB tubes at 35 ± 2°C for 26 ± 2 h.
19
1.14.3
Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of
20
the enriched sample over the surface of the plate. Incubate the MOX at 35
21
± 2°C for 26 ±2 h.
22
1.14.4
Examine the MOX plates for colonies with morphology typical of
Listeria
23
spp
.
At 26 ± 2 h, suspect colonies are typically small (ca. 1 mm) and are
24
surrounded by a zone of darkening due to esculin hydrolysis.
25
1.14.5
If suspect colonies are present on MOX, transfer suspect colonies to HL
26
agar.
27
1.14.6
If no suspect colonies are evident, re-incubate the MOX plate for an
28
additional 26 ± 2 hour.
29
1.14.7
After 26 ± 2 h of incubation, examine the FB for the potential presence of
30
Listeria spp.,
by visual examination of the broth for darkening due to
31
esculin hydrolysis.
32
1.14.8
If any degree of FB darkening is evident, aseptically dispense a drop
33
approximating 0.1 ± 0.02 ml of FB onto a MOX plate. Swab or streak 25-
34
40% of the surface of the MOX plate with the FB inoculum. Use a loop to
35
streak for isolation from the initial swab/streak quadrant onto the
36
remainder of the plate. Incubate the MOX plate at 35 ± 2°C for 26 ± 2 h.
37
1.14.9
If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a total
38
incubation time of 48 ± 2 h has been achieved.
39
1.14.10Re-examine the FB for evidence of darkening after 48 ± 2 h of total
40
incubation. If any degree of darkening is evident, swab, streak and
41
incubate a MOX plate.
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1.14.11If no darkening of FB is evident and no suspect MOX and/or HL colonies
43
have been demonstrated, the sample is considered negative for
Listeria
44
spp.
45
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol
OMA ERP - June 2016
ERP Use Only