AOAC ERP MICRO AUGUST 2018

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

The inclusivity and exclusivity study examines the ability of the Peel Plate EB method to detect a variety 1 of Enterobacteriaceae (EB) strains and to distinguish those strains from closely related non-EB strains 2 and species. Ideally, at least 50% of the organisms tested should be foodborne isolates.

3 4 5 6 7 8 9

4.1

For inclusivity, select at least 50 different isolates of EB strains (Table 1).

4.2 Culture each strain in nutrient broth for 37 ± 1°C for 24 ± 2 h. Dilute each strain in sterile

Butterfield’s buffer so that the inoculum level is 10–100 times greater than the

minimum level for quantification (approximately 100 CFU/mL).

4.3 For exclusivity, select at least 30 isolates of closely related non-EB species and strains

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(Table 2).

4.4 Culture exclusivity strains in non-selective media (such as BHI) at 37 ± 2°C for 20–24 h. Dilute each strain in sterile Butterfield’s buffer so that the inoculum is approximately

100–200 CFU/mL.

4.5 Blind-code and randomize inclusivity and exclusivity isolates so that the analyst does not

know the identity of the test samples.

4.6 Place 1 mL of sample suspension from appropriate dilutions onto center of Peel Plate EB medium. Re-apply adhesive cover. Press around edges of plate to insure proper seal. Incubate at 37 ± 1°C. Record results after 24 and 48 h of incubation. View through the

clear side of plate. Each spot represents 1 CFU of EB, regardless of color.

4.7

Decode and tabulate results by strain.

4.8 Report inclusivity data as the number of strains detected out of the total number tested. For example, “Of the 50 coliform inclusivity strains tested, 48 were detected and 2 were

not detected. Those strains not detected were…”

4.9 Report exclusivity as the number of exclusivity organisms not detected out of the total number tested. For example, “Of the 30 exclusivity strains tested, 29 were not detected

and 1 was detected. The strain detected was…”

4.10 Approximately 50% of the strains should be of food origin. Report strain, subgroup, serogroup, source and origin in the inclusivity and exclusivity tables where applicable. ERP Use Only Table 1. Proposed Enterobacteriaceae Inclusivity Strains Genus species Subspecies Source Origin feces amalonaticus Not Available clinical isolate, California

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Number

1 2 3 4 5 6 7 8 9

Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter Cronobacter Cronobacter Cronobacter Cronobacter Cronobacter Cronobacter Cronobacter Edwardsiella Enterobacter

ATCC a 25405 ATCC 27156 ATCC 43162 ATCC 51633 QL b 100813-2A DSM c 18706 DSM 27966 CCUG d 66106 CCUG 28859 DSM 21870 DSM 19145 CCUG 28863 ATCC 15947 ATCC 35029

koseri braakii farmeri freundii

human feces

sliced deli meat (turkey)

dublinensis condimenti helveticus malonaticus muytjensii

Infant Formula Infant Formula Product industry Product industry Product industry Formula

10 11 12 13 14

pulveris sakazakii

Human cerebrospinal fluid

tarda

feces, human Not Available

aerogenes