AOAC ERP MICRO AUGUST 2018

OMAMAN-44 A: Collaborative Study Manuscript Expert Review Panel Use Only August 2018

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Collaborative Study Test Portion Analysis

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Collaborators followed the appropriate preparation and analysis protocol provided to them in the

collaborator instructions (Version 3, February 2018). Each collaborator received 8 test portions (2 high, 2 436

medium, 2 low and 2 uninoculated). A 25 g test portion was diluted with 225 mL of BPW (ISO) and 437

homogenized with a paddle blender for 2 m ± 10 s. Ten-fold serial dilutions of each sample were 438

prepared and a 1.0 mL aliquot of each dilution was plated onto a single Peel Plate EB for each dilution. 439

Plates were incubated at 37 ± 1 o C for 24 h and enumerated. After enumeration, plates were re- 440

incubated at 37 ± 1 o C for an additional 24 h (48 hours total). Plates were re-enumerated at 48 h. Each 441

spot on the plate represented an EB colony and was enumerated. Plates containing greater than 150 442

colonies were recorded as too numerous to count. Final results were determined by multiplying the 443

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counts by the dilution factor for that plate.

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Each test portion analyzed by the Peel Plate EB method was also analyzed using either the ISO

21528-1 or 21528-2 reference method in a paired study design. The un-inoculated and low test portions 446

were analyzed via the ISO 21528-1 reference method and the medium and high level samples were 447

analyzed via the ISO 21528-2 reference method. For ISO 21528-1, a 3-tube MPN was prepared. Positive 448

tubes, those showing turbidity indicating growth, were struck to VRBG for visual determination of typical 449

colonies (red to purple with or without zones of precipitate). For ISO 21528-2, serial dilutions for each 450 ERP Use Only sample were plated in duplicate using VRBG. Agar plates were incubated for 24 ± 2 h at 37 ± 1 o C. Typical 451 colonies in the countable range (<150) were enumerated using a standard colony counter. For both 452 parts 1 and parts 2, typical colonies were confirmed positive for Enterobacteriaceae by a spot oxidase 453

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test and glucose agar test.

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