AOAC-RI ERP Micro June 2016

OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol OMA ERP - June 2016 ERP Use Only

3M MDA 2 Listeria species and L. monocytogenes Collaborative Study OMA-2016-MONTH-XXX

1.6 Transfer 20 µL Negative Control [NC], sterile enrichment medium (e.g. Demi- Fraser Broth) into a Lysis Solution [LS] tube after all enriched samples have been

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completed. Do not use water as a NC.

1.7 Place uncovered LS tubes in 3M MDS Heat block, heat 15±1 min at 100±1°C.

LS Solution will change from pink (cool) to yellow (hot).

1.8 Place LS tubes (without rack lid) in chill block insert (at ambient temperature) for

5-10 min. Lysis solution in LS tube will revert to pink color.

1.9 Transfer 20 µL sample lysate from the upper portion of fluid in the LS tube into regent tube. Mix gently by pipetting up and down 5 times. 1.10 Transfer 20 µL of NC lysate into a reagent tube. 1.11 Transfer 20 µL of NC lysate into a Reagent Control (RC) tube. 1.12 Load capped tubes into speed loader tray and close lid.

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1.13 Start assay.

1.14 Validation Study Confirmation

1.14.1 Confirm each test portion, regardless of presumptive result. 1.14.2 Transfer 0.1 ± 0.02 ml of the enriched sample to 10 ± 0.5 ml of Fraser Broth (FB). As per media preparation instructions, be sure that appropriate supplements have been added to the FB prior to inoculation. 1.14.3 Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of the enriched sample over the surface of the plate. Incubate the MOX at 35 1.14.4 Examine the MOX plates for colonies with morphology typical of Listeria spp . At 26 ± 2 h, suspect colonies are typically small (ca. 1 mm) and are surrounded by a zone of darkening due to esculin hydrolysis. 1.14.5 If suspect colonies are present on MOX, transfer suspect colonies to HL 1.14.7 After 26 ± 2 h of incubation, examine the FB for the potential presence of Listeria spp., by visual examination of the broth for darkening due to AOAC Research Institute Expert Review Panel Use Only esculin hydrolysis. 1.14.8 If any degree of FB darkening is evident, aseptically dispense a drop approximating 0.1 ± 0.02 ml of FB onto a MOX plate. Swab or streak 25- 40% of the surface of the MOX plate with the FB inoculum. Use a loop to streak for isolation from the initial swab/streak quadrant onto the remainder of the plate. Incubate the MOX plate at 35 ± 2°C for 26 ± 2 h. 1.14.9 If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a total Incubate inoculated FB tubes at 35 ± 2°C for 26 ± 2 h. ± 2°C for 26 ±2 h. agar. 1.14.6 If no suspect colonies are evident, re-incubate the MOX plate for an additional 26 ± 2 hour. 1.14.10Re-examine the FB for evidence of darkening after 48 ± 2 h of total incubation. If any degree of darkening is evident, swab, streak and 1.14.11If no darkening of FB is evident and no suspect MOX and/or HL colonies have been demonstrated, the sample is considered negative for Listeria incubate a MOX plate. incubation time of 48 ± 2 h has been achieved.

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5/4/16