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

1.18 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. Incubate inoculated FB tubes at 35 ± 1.19 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 ± 2°C for 1.20 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 1.21 If suspect colonies are present on MOX, transfer suspect colonies to HL agar. 1.22 If no suspect colonies are evident, re-incubate the MOX plate for an additional 26 1.23 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 esculin hydrolysis. 1.24 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. 1.26 Re-examine the FB for evidence of darkening after 48 ± 2 h of total incubation. If any degree of darkening is evident, swab, streak and incubate a MOX plate. 1.27 If no darkening of FB is evident and no suspect MOX and/or HL colonies have been demonstrated, the sample is considered negative for Listeria spp. 1.28 If suspect colonies are present on MOX from any source, streak for isolation on one or more HL agar plates. Incubate the streaked HL at 35 ± 2°C for 22 ± 4 h. 1.29 After incubation, examine the HL plate(s) against backlight for translucent 1.30 If at least one suspect colony is clearly isolated, proceed to confirmatory testing. Hold all HL plates containing suspect colonies (room temperature or refrigeration) until confirmatory testing is complete. AOAC Research Institute Exp rt Review Panel Use Only 1.31 If suspect colonies or β-hemolytic growth are present on HL but not clearly isolated, re-streak representative suspect colonies/growth onto one or more fresh HL plates and incubate at 35 ± 2°C for 22 ± 4 h. 1.32 Confirm ALL test portions according to the USDA FSIS biochemical/serological procedures. Biochemical tests will be performed using API or VITEK 2 GP. Please follow manufacturer’s instructions when using any of these rapid methods. 2°C for 26 ± 2 h. 26 ±2 h. zone of darkening due to esculin hydrolysis. ± 2 hour. Incubate the MOX plate at 35 ± 2°C for 26 ± 2 h. 1.25 If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a total incubation time of 48 ± 2 h has been achieved. colonies surrounded by a small zone of β-hemolysis.

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Final Results 42 43 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

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