Microbiology Methods for ERP Review 3-2020

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GENE-UP SLM Matrix Extension

June 19, 2017

incubated at 35 ± 2 o C for 18 ‐ 24 h. Growth from the TSA slant was used to conduct the polyvalent O 1 serological test for biochemical confirmation and final confirmations using VITEK ® 2 GN following AOAC 2 Official Method 2011.17. 3 4 FDA ‐ BAM Chapter 5 Detection and Enumeration of Salmonella 5 Following the FDA ‐ BAM Ch. 5 method, powdered egg, whey protein, pet food and peanut butter 25 g test 6 portions were combined with 225 mL lactose broth then incubated 24 ± 2 h at 35°C. Instant nonfat dried 7 milk 25 g test portions were combined with 225 mL brilliant green water and incubated for 24 ± 2 h at 35°C. 8 Dark chocolate 25 g test portions were combined with 225 mL reconstituted nonfat dry milk and incubated 9 24 ± 2 h at 35°C. Garlic powder 25 g test portions were combined with TSB plus K 2 SO 3 (5 g K 2 SO 3 per 1000 10 mL TSB, resulting in final 0.5% K 2 SO 3 concentration) and incubated 24 ± 2 h at 35°C. A 0.1 mL aliquot from 11 the primary enrichment was added to 10 mL RV broth (prepared from individual ingredients) and a 1 mL 12 aliquot was added to 10 mL TT broth then incubated at 42  0.2°C for 24  2 h. After aerobic plate count 13 evaluation for each product, TT broth for high microbial load products were incubated at 43 ± 0.2  C for 24 14  2 h in a circulating water bath. TT broth for low microbial load products were incubated at 35 ± 2.0  C for 15 24  2 h in a forced air incubator. A 3 mm loopful (10  L) of each RV broth and TT broth were streaked onto 16 bismuth sulfite (BS), XLD and Hektoen enteric (HE) agars and incubated at 35  C for 24  2 h. Typical 17 colonies were transferred to TSI and LIA. Incubate at 35  C for 24  2 h. At least one typical colony per test 18 portion was confirmed with biochemical/serological procedures prescribed by the FDA ‐ BAM method. The 19 VITEK® GNI (Official Method 991.13) was used as an alternative to the conventional biochemical tests. The 20 somatic (O) and flagellar (H) tests were also be performed. 23 The level of Salmonella in the low level inoculum for all 25 g test portions was determined by Most 24 Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x 25 g (reference method test 25 portions), and 5 x 10 g inoculated test samples. The level of Salmonella in the high level inoculum for all 25 26 g test portions was determined by MPN on the day of analysis by evaluating 5 x 25 g (reference method 27 test portions), 5 x 10 g, and 5 x 1 g inoculated test samples. 28 29 The level of Salmonella in the low level inoculum for all 100 g test portions was determined by MPN on the 30 day of analysis by evaluating 5 x 200 g, 20 x 100 g (reference method test portions), and 5 x 25 g inoculated 31 test samples. The level of Salmonella in the high level inoculum for all 100 g test portions was determined 32 by MPN on the day of analysis by evaluating 5 x 100 g (reference method test portions), 5 x 25 g, and 5 x 10 33 g inoculated test samples. The test portion size for the MPN of each matrix is presented below in Table B. 34 Each test portion was enriched with the reference method enrichment and analyzed by the reference 35 method procedure. The number of positives from the 3 test levels was used to calculate the MPN using the 36 LCF MPN calculator version 1.6 (6). 37 38 The probability of detection (POD) was calculated as the number of positive outcomes divided by the total 39 number of trials. The POD was calculated for the candidate presumptive results, POD CP, the candidate 40 confirmatory results, POD CC , the difference in the candidate presumptive and confirmatory results, dPOD CP, 41 the reference method, POD R , and the difference in the confirmed candidate and reference methods, 42 dPOD C . POD analyses were conducted for the GENE UP Salmonella and for the reference methods. 43 44 Chicken carcass rinsate : Chicken carcass rinsate had >2500 CFU/g of background organisms and was 45 negative for natural occurring Salmonella . Analysis was performed on three levels of S.enterica serovar 46 Abeatetuba inoculation: 0 and 1.1 and 1.5 CFU/carcass for the GENE ‐ UP and the MLG reference methods. 47 The un ‐ inoculated samples resulted in 0 positives out of 5 replicates for both methods. The 1.5 CFU level 48 samples resulted in 5 positives out of 5 replicates for both methods. For the 1.1 CFU level of inoculation, 49 21 22 Most Probable Number

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