Microbiology Methods for ERP Review 3-2020

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GENE-UP SLM Matrix Extension

June 19, 2017

Creamy peanut butter (375 g) 1 Creamy peanut butter was inoculated with Salmonella enterica subsp. Potsdam. A 375 g test portion was 2 homogenized with 1125 mL of pre ‐ warmed (42°C) BPW and incubated at 41.5 ‐ 42°C for 20 ‐ 24 h. 5 Dark chocolate was inoculated with Salmonella enterica subsp. Cerro. A 375 g test portion was 6 homogenized with 3375 mL of pre ‐ warmed (42°C) BPW and incubated at 41.5 ‐ 42°C for 20 ‐ 24 h. 9 Garlic powder was inoculated with Salmonella enterica subsp. Arizonae. A 375 g test portion was 10 homogenized with 3375 mL of pre ‐ warmed (42°C) BPW + K 2 SO 3 and incubated at 35°C for 22 ‐ 26 h. 11 12 After incubation, samples were analyzed according to the GENE ‐ UP Salmonella method instructions. The 13 required number of GENE ‐ UP lysis tubes were placed in the GENE ‐ UP Lysis Tube Rack. The GENE ‐ UP Lysis 14 Rack was placed on the GENE ‐ UP Heavy Rack. From each enriched sample, 15 μ L was added to separate 15 lysis tubes. The GENE ‐ UP Lysis Rack was then moved to the GENE ‐ Up Lysis Rack Adaptor. The bead beater 16 was run at maximum speed for 5 min, with a speed above 2000 rpm. The GENE ‐ UP Lysis Rack was placed 17 back into the GENE ‐ UP Heavy Rack Holder to proceed with final set up for PCR. 18 19 Each reagent vial was tapped on the bench prior to removing the rubber cap. Assuring the pellet was at or 20 near the bottom of the vial, 45 μ L of reconstituted buffer was added and mixed. A 5 μ L aliquot of this 21 solution was added into a PCR strip tube in the GENE ‐ UP PCR Rack. Using a 10 μ L BioTix filter pipette tip, 5 22 μ L of lysed sample was added to the corresponding PCR tube. The PCR strip tubes were sealed with the 23 strip caps and centrifuged for 10 seconds then analyzed with the GENE ‐ UP instrument. 24 25 All enrichments, regardless of presumptive results, were confirmed following an alternative protocol, ASAP 26 agar and the appropriate reference method, starting with section D, Isolation of Salmonella in Chapter 5 of 27 the FDA ‐ BAM and section 4.6, Selective Enrichment and Plating Media in version 4.09 of the USDA/FSIS 28 MLG. 29 30 USDA/FSIS MLG 4.09 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and 31 Catfish Products 32 For the USDA/FSIS MLG 4.09 reference method, carcass rinsate 30 mL test portions were combined with 30 33 mL of BPW, mixed and incubated at 35  2  C for 20 ‐ 24h. Fresh raw chicken breast 25 g test portion was 34 combined with 225 mL of BPW, homogenized for 2 min and incubated at 35  2  C for 20 ‐ 24h. Beef trim, 35 ground pork and ground bison 25 g test portions were each combined with 75 mL of mTSB, homogenize for 36 2 min and incubated at 42  1  C for 15 ‐ 24h. Pasteurized liquid egg 100 g test portions were each combined 37 with 900 mL of BPW, mixed and incubated at 35  2  C for 18 ‐ 24h. 38 39 After incubation, 0.1 ± 0.02 mL of each sample was transferred to 10 mL of modified Rappaport ‐ Vasilliadis 40 broth (mRV) and 0.5 ± 0.05 mL into 10 mL of tetrathionate Hajna broth (TT Hajna). The broths were 41 incubated in a water bath at 42 ± 0.5°C for 18 ‐ 24 h. Following incubation and for each sample, a loopful 42 from each broth replicate was streaked to xylose ‐ lysine ‐ tergitol™ 4 (XLT4) and brilliant green sulfa agar 43 (BGSA). Both selective agars were incubated at 35 ± 2 o C for 18 ‐ 24 h. Presumptive positive Salmonella 44 colonies from each selective agar were picked and transferred to triple sugar iron agar (TSI) and lysine iron 45 agar (LIA) slants and incubated at 35 ± 2°C for 24 ± 2 h. Growth from samples producing typical biochemical 46 reactions in TSI was transferred to trypticase soy ‐ tryptose broth (TSTB) and incubated at 35 ± 2°C for 24 ± 2 47 h. Growth from the TSTB was used to conduct the flagellar H serological test. Growth from samples 48 producing typical biochemical reactions in TSI and LIA, were streaked to trypticase soy agar (TSA) slants and 49 3 4 Dark chocolate (375 g) 7 8 Garlic powder (375 g)

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