ERP Micro December 2019

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3.2.93.2.10

GDS O157 Method

3.2.9.13.2.10.1 All steps should be performed as indicated in the GDS O157

Formatted: Indent: Left: 1.5", Hanging: 0.63", Outline numbered + Level: 4 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1.5" + Indent at: 2"

package insert.

3.2.9.23.2.10.2 For25 g sample enrichment, add 225 mL pre-warmed (41–43°C) mEHEC media to each 25 g test portion. For 375 g sample enrichment, add 1,500 mL pre-warmed (41–43°C) mEHEC media to each 375 g test 3.2.9.33.2.10.3 Incubate 25 g samples for 6.5 – 18 h and 375 g samples for 8–18 h at 41–43°C. For frozen samples, incubate 25 g samples for 8 – 18 h and 375 g samples for 10–18 h at 41–43°C. For green onions, incubate 3.2.9.43.2.10.4 Conduct sample pp reparation and test procedure according to the Assurance E . coli O157:H7 Tq directions for use ( Attachment 1 ). 3.2.9.53.2.10.5 Confirm GDS O157 enrichments for E . coli O157:H7 : portion. Homogenize sample by hand or macerate for 2 min. for 25 g samples for 8 – 18 h at 41–43°C.

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Formatted: Font: Not Bold Formatted: Indent: Left: 2.13", Outline numbered + Level: 5 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 2" + Indent at: 2.75"

Reference method confirmation: For beef trim,

3.2.9.5.13.2.10.5.1

ground beef and FTB enriched test portions, conduct immunomagnetic separation and culture plating for isolation and confirmation as described in MLG 5 .09C.00 . Confirm latex-positive colonies with Real-time PCR, agglutination, and biochemical identification. Any OMA approved biochemical test can be used for rapid

biochemical confirmations.

3.2.9.63.2.10.6 Report presumptive and confirmed results for each GDS O157

Formatted: Indent: Left: 1.5", Hanging: 0.63", Outline numbered + Level: 4 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1.5" + Indent at: 2" Formatted: Outline numbered + Level: 3 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1" + Indent at: 1.5" Formatted: Indent: Left: 1.5", Hanging: 0.63", Outline numbered + Level: 4 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1.5" + Indent at: 2"

enriched test portion for each confirmation method.

3.2.103.2.11

MLG 5 .09C.00

3.2.10.13.2.11.1

To each 3 25 g raw beef trim or FTB test portion, add 975

225 ± 19.5 mL Modified Tryptone Soya Broth (mTSB). Pummel, blend, or hand massage until well mixed. Incubate at 42 ± 1°C for 15–24 h.

3.2.10.23.2.11.2

Continue with IMS, acid wash treatment, and plating

Formatted: Indent: Left: 1.5", Hanging: 0.63", Outline numbered + Level: 4 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1.5" + Indent at: 2"

onto mRBA as described in MLG 5.6.1. Incubate plates for 20–24 h at

35 ± 2°C.

.

3.2.10.33.2.11.3

Examine plates for colonies that agglutinate with latex

Formatted: Indent: Left: 1.5", Hanging: 0.63", Outline numbered + Level: 4 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 1.5" + Indent at: 2"

agglutination reagents specific for the serogroup O157. Streak all agglutination-positive colonies, up to five total from each sample, onto

SBA plates. Incubate SBA plates for 16–24 h at 35 ± 2°C.

3.2.10.43.2.11.4

Examine SBA plates for purity. If plates appear pure and

uncontaminated, perform confirmatory tests

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Perform confirmations with the following tests:

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