AOAC ERP MICRO AUGUST 2018

OMAMAN-30 D/ PTM Validation Report 081501 OMA ERP - June 2016 ERP Use Only

plates were examined for hemolysis. Final confirmation was conducted using the VITEK ® GP

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Biochemical Identification card following AOAC OMA 2013.02.

AOAC 993.12 Listeria Reference Method

Twenty-five gram test portions were enriched in 225 mL ± 5 mL of selective enrichment medium, containing yeast extract (1.35 g/225 mL), acriflavine mono hydrochloride (1.125 g/225 mL), nalidixic acid (sodium salt) solution (1.125 g/225 mL), and cycloheximide (2.25 g/225 mL), and incubated at 30 ± 1 °C for 48 hours. The enriched samples were then streaked to Oxford Agar (OXA) and incubated at 37 ± 1°C for 48 hours. At 48 hours, the OXA plates were examined for suspect colonies. If present, up to 5 suspect colonies were streaked to TSA/YE to obtain well isolated, pure colonies. Pure colonies on TSA/YE were analyzed for Gram-stain reaction and for catalase activity. Catalase positive, Gram positive rods colonies were then stabbed to SBA and incubated at 37 ±1 °C for 48 hours. After 48 hours, the SBA plates were examined for typical hemolytic reactions. The same colony picked to SBA was also transferred to TSB/YE. The TSB/YE cultures were incubated at 25 ± 1 °C overnight, or until the broth was turbid, indicating sufficient growth. Another TSB/YE tube was inoculated with the same colony and incubated at 37 ±1 °C for 24 hours to be used for carbohydrate utilization testing. The TSB/YE tube incubated at 25 ±1 °C were used to prepare a wet mount slide to determine motility pattern. From the TSB/YE tube incubated at 37 ± 1 °C, Motility Test Medium (MTM) was stabbed and incubated at 25 ±1 o C. After 2 days, and up to 7 days, the MTM tubes were observed for umbrella-like growth. Additionally from the TSB/YE tube, a loopful (0.1 mL) was transferred into carbohydrate fermentation broth (purple broth) containing 1.0 mL of either 5% rhamnose or 5% xylose. The purple broth tubes were incubated at 37 ± 1 °C and examined for up For the ISO 11290-1/A1 reference method, 25 g test portions were enriched with 225 half Fraser broth. All test portions were mechanically stomached for two minutes. The test portions were incubated at 30 ± 1°C for 24 ± 3 hours. After incubation, 0.1 mL of the sample enrichment was transferred to 10 mLFB containing 0.1 mL of 5% ferric ammonium citrate and incubated at 37 ± 1°C for 48 ± 3 hours. After 48 ± 3 hours, a loopful of the sample secondary FB enrichment was streaked to PALCAM and OAA (Ottovani-Agosti Agar )and incubated at 37 ± 1°C for 24 ± 3 hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAAand incubated at 37 ± 1°C for 24 ± 3 hours. PALCAM and OAA agar plates were examined for the presence of suspect colonies. If no suspect colonies were present, the agar plate was re-incubated for an additional 18-24 hours at 37 ± 1°C. If no suspect colonies present the sample was determined to be negative for Listeria. If suspect colonies were present on the PALCAM or OAA agar plates, these suspect colonies were streaked to HBO and incubated at 37 ± 1 o C for 18- 24 hours. HBO plates were examined for hemolysis reactions and well-isolated colonies were transferred to BHI broth and incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and examined for morphology by preparing a Gram stain. Additionally, purified HBO isolates were identified using the VITEK ® GP Biochemical Identification following AOAC OMA 2013.02. AOAC Research Institute Expert Review Pane Use Only to 7 days. ISO 11290-1/A1 Reference Method

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