AOAC ERP MICRO AUGUST 2018

OMAMAN-44 B: Collaborative Study Protocol Expert Review Panel Use Only August 2018

5.6.5 Count all pink to red or purple colonies (with or without precipitation halos). Countable range is <150. Average the duplicate results. Multiply by dilution factor and report as total EB count/g, mL, carcass or 100 cm 2 of product. 5.6.6 For confirmation, select 5 colonies from each replicate (25 from each spike level) and streak to TSA. Incubate TSA at 37 ± 1°C for 24 ± 2 h. Conduct a spot oxidase test. Stab oxidase negative colony into Glucose OF Medium. Add an overlay of mineral oil and incubate tubes at 37 ± 1°C for 24 ± 2 h. If a yellow

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color develops, the reaction is regarded as positive.

5.7

ISO 21528-1:2017 (Low levels of contamination; < 100 CFU/g or mL)

5.7.1 Milk, non-fat dry milk, butter, infant formula, infant cereal, vanilla ice cream : Aseptically weigh 25 ± 1 g sample into 225 mL of BPW (ISO). Stomach for 2 min ±

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15 s.

5.7.2 Carcass rinse : Add 400 mL of BPW (ISO) to the carcass bag, approximately one- half the volume into the interior cavity and the other half over the skin. Rinse inside and out with a rocking motion for 1 min ± 10 s at a rate of approximately 5.7.3 Environmental surface sponge sample (stainless steel) : Moisten the sampling sponge with 10 mL diluent. Sample each 100 cm 2 area in a horizontal and vertical motion. Return sponge to bag. Hold sponge at room temperature for at least 2 h prior to analysis. Add 25 mL of BPW (ISO) diluent and homogenize for 1 5.7.4 From each sample type, transfer 10 mL of the 1:10 food dilution, carcass rinse, or sponge sample into each of 3 test tubes (10 -1 ); transfer 1 mL of the 1:10 dilution into each of 3 x 9 mL test tubes of BPW (ISO) (10 -2 ), and transfer 1 mL of the 10 -2 dilution into each of 3 x 9 mL test tubes of BPW (ISO) (10 -3 ). Incubate tubes at 37 ± 1°C for 18 ± 2 h. Streak all tubes onto plates containing VRBG and ERP Use Only incubate at 37 ± 1°C for 24 ± 2 h. 5.7.5 Select up to 5 characteristic colonies and streak to TSA. Incubate TSA at 37 ± 1°C for 24 ± 2 h. Conduct a spot oxidase test. Stab oxidase negative colony into Glucose OF Medium. Add an overlay of mineral oil and incubate tubes at 37 ± 1°C for 24 ± 2 h. If a yellow color develops, the reaction is regarded as positive. Data Analysis 5.8.1 Each matrix and time point must be analyzed separately using duplicate paired average or singlet result compared to reference paired average. 5.8.2 Perform a logarithmic transformation on the reported CFU/g: 35 forward and back swings per minute. min ± 10 s.

5.8

Log10[CFU/g + (0.1)f]

Where f is the reported CFU/unit corresponding to the smallest reportable

result. See Annex H of Appendix J (1).

5.8.3 Perform outlier tests (Cochran and Grubbs). Remove outliers from data analysis

only if there is a justifiable cause.

5.8.4 Plot the candidate method result (x-axis) vs. the reference method result (y-

axis). Usually major discrepancies will appear.