ERP Micro December 2019

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a) biochemical identification (BioMerieux API 20E or VITEK® 2).

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b) latex agglutination for O157 and H7 antigens.

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c) shiga toxin/toxin genes confirmation either by toxin assay or detection of one

or more toxin genes by PCR.

3.2.12 BAM Chapter 4A

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3.2.12.1 To each 25 g green onionsfood, add 225 mL Modified Buffered Peptone water with pyruvate (mBPWp). Blend or stomach until well mixed. Incubate at 37 ± 1°C for 5 h. Add 1 mL each of the Acriflavin-Cefsulodin- Vancomycin (ACV) supplements per 225 mL of mBPWp and incubate 42

± 1°C overnight (18 – 24 h).

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3.2.12.2 Continue plating onto TC-SMAC and one chromogenic agar (Rainbow Agar O157) as described in BAM Chaahpter 4A Section P. Incubate

plates for 18–24 h at 37 ± 1°C.

3.2.12.3 Examine plates for colonies that agglutinate with latex agglutination reagents specific for the serogroup O157. Streak all agglutination- positive colonies, up to ten total from each sample, onto TSA-YE plates.

Incubate TSA-YE plates for 18–24 h at 37 ± 1°C.

3.2.12.4 Examine TSA-YE plates for purity. If plates appear pure and

uncontaminated, perform confirmatory tests.

Perform confirmations with the following tests:

a) biochemical identification (BioMerieux API 20E or VITEK® 2).

b) latex agglutination for O157 and H7 antigens.

3.2.113.2.13

MPN Procedure

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3.2.11.13.2.13.1

Perform a 5-tube, 3-level MPN on the low and high

inoculation levels of each food matrix. Prepare 5 test portions of 50 g, 5 test portions at 25 g and 5 test portions of 10 g at each inoculation level. To the 50 g test portions, add 450 mL of the reference method enrichment broth. To the 25-g test portions, add 225 mL of the reference method enrichment broth. To the 10 g test portions, add 90 mL of the reference method enrichment broth. Follow the appropriate reference method for the matrix through to confirmations. used as part of the MPN analysis. For beef matrices, low (fractional) inoculation level, use the twenty 325 g test portions as the middle level. Include five 650 g (50 g low level inoculated sample plus 600 g Alternatively, the reference method replicates may be

3.2.11.23.2.13.2

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