AOAC ERP MICRO AUGUST 2018

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

of n-BPW. Test each replicate in duplicate and marking one the two duplicates

1 2 3 4 5 6 7 8 9

with an S to represent a singlet result.

5.5.6 Environmental surface sponge sample (stainless steel) : Moisten the sampling sponge with 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 peptone water diluent and homogenize for 1 min. Prepare all decimal dilutions by mixing 10 mL previous dilution with 90 mL 5.5.7 Lift the Peel Plate tab and pull adhesive cover to fully expose the dried culture disc. Keeping the plate flat, vertically dispense 1 mL of sample suspension onto the center of disc. Re-apply adhesive cover. Press around edges of plate to insure proper seal. Test each replicate in duplicate and marking one the two 5.5.8 Incubate plates in the dark with the clear side up, at 37 ± 1°C for 24–48 ± 3 h). 5.5.9 Count all colonies and record results at 24 and 48 h. The suitable colony counting range is 1–150 CFU/mL. Report each duplicate result as the singlet result and the duplicate result which will be used to determine an average. Multiply each by dilution factor and report as total EB count/g, mL, carcass or 5.5.10 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 ISO 2158-2:2017 (medium to high levels of contamination; >100 CFU/g or mL) 5.6.1 Milk, non-fat dry milk, butter, infant formula, infant cereal, vanilla ice cream : Use same food dilution as prepared for Peel Plate EB, e.g. weigh 25 ± 1 g sample into 225 mL 0.1% Peptone Water (PW). Stomach 2 min ± 15 s (See 5.5.3 and 5.5.4). Perform further dilutions by transferring 1 mL of the initial suspension into a 9 mL sterile dilution blank. Dilute portions to obtain 1–150 CFU/plate. 5.6.2 Carcass rinse : Prepare carcass rinse as in 5.5 using 400 mL of Buffered Phosphate Diluent (BPD) to the carcass bag, approximately one-half the volume ERP Use Only into the interior cavity and the other half over the skin. Perform further dilutions by transferring 1 mL of the initial suspension into a 9 mL sterile dilution of peptone water diluent or equivalent. duplicates with an S to represent a singlet result. 100 cm 2 of product. color develops, the reaction is regarded as positive.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

5.6

blank. Dilute portions to obtain 1–150 CFU/plate.

5.6.3 Environmental surface sponge sample (stainless steel) : Use the same surface sponge samples as in 5.5.6. Prepare all decimal dilutions by mixing 10 mL

previous dilution with 90 mL of peptone water diluent.

5.6.4 Plate 1 mL of each dilution in duplicate. Pour 10 mL of tempered VRBG agar to each plate. Mix well. After completely solidified, pour overlay of approximately 8–15 mL VRBG. Invert when solidified. Incubate plates at 37 ± 1°C for 24 ± 2 h.