AOAC-RI ERP Micro June 2016

OMAMAN-29 D/ PTM Validation Report 111501 OMA ERP - June 2016 ERP Use Only

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TSA/YE plates were incubated at 35 ± 1°C for 24-48 hours and then examined for purity. Pure colonies were tested for catalase reactivity and a Gram Stain was conducted. A pure Listeria colony was transferred to Trypticase Soy Broth containing 0.6% yeast extract (TSB/YE). The TSB/YE cultures were incubated at 25 ± 1°C overnight, or until the broth was turbid, indicating sufficient growth. Catalase-positive organisms were stabbed into plates of 5% Sheep Blood Agar (SBA) and incubated at 35 ± 1°C for 24-48 hours. The TSB/YE tubes incubated at 25 ±1°C were used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA plates were examined for hemolysis. Final confirmation was conducted using the VITEK ® GP 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 was 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 mL FB 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 Ottovani-Agosti Agar (OAA) and incubated at 37 ± 1°C for 24 ± 3 hours. A loopful of the sample primary enrichment was also streaked to PALCAM and OAA and 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 Biochemical Identification card following AOAC OMA 2013.02. AOAC 993.12 Listeria Reference Method to 7 days. ISO 11290-1/A1 Reference Method

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AOAC Research Institute Expert Review Panel Use Only