United States Food Safety Office of Laboratory QA Staff

Department of and Inspection Public Health 950 College Station Road

Agriculture Service Science Athens, GA 30605

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Laboratory Guidebook
Notice of Change

Chapter new, revised, or archived: MLG 5 Appendix 1.02

Title: Flow Chart Specific for FSIS Laboratory Escherichia coli O157:H7 Analysis

Effective Date: 06/29/14

Description and purpose of change(s):

This flow chart was issued in association with MLG 5.08 Detection, Isolation and
Identification of Escherichia coli O157:H7 from Meat Products and Carcass and
Environmental Sponges and MLG 5A.04 FSIS Procedure for the Use of Escherichia
coli O157:H7 Screening Tests for Meat Products and Carcass and Environmental
Sponges.

The methods described in this guidebook are for use by the FSIS laboratories. FSIS does not
specifically endorse any of the mentioned test products and acknowledges that equivalent
products may be available for laboratory use. Method validation is necessary to demonstrate the
equivalence of alternative tests as detailed in the document titled “FSIS Guidance for Evaluating
Test Kit Performance” available on the FSIS website.
_______________________________________________________________________________________
QD-F-Micro-0004.07 Issuing Authority: Director, Laboratory Quality Assurance Staff (LQAS)
Page 1 of 1
Effective: 09/30/13
 Flow Chart Specific for FSIS Laboratory Escherichia coli O157:H7 Analysis
Per 325g sample add 975ml of mTSB1, stomach for 2 min. For sponges with 10 ml of
buffer add 50 ml mTSB1 to bring the volume to 60 ml, mix well. * This chart represents the best
Day 1

Incubate @ 42 ± 1°C for 15 to 24 hrs. case scenario but analyses may

take longer due to normal
analytical circumstances such
NOTE: Sample receipt temperature of ≤15oC is required. as restreaking isolates for
purity.

Lateral (-)
BAX®
RT Alternative Flow Report as
PCR Device NEGATIVE
Day 2

(+) Report as Potential (+)

Streak prepared mTSB1 to mRBA2 (2 dilutions)

Prepare aliquot of mTSB1
for Immunomagnetic then incubate @ 35 ± 2°C for 20-24 hrs.
bead (IMS) capture Perform acid treatment step and streak prepared mTSB1 to
mRBA2 (2 dilutions) then incubate @35 ± 2°C for 20-24 hrs.

Pick typical well-isolated colonies from mRBA and test for the O157 antigen using
the RIM4 latex agglutination test.
Day 3

(+) Report as Presumptive (+)

(-)
Report as
Pick up to five latex positive colonies and streak for
NEGATIVE purity to SBA4 and incubate @35°C for 16-24hrs.

4
Examine SBA under UV light for contamination and test a colony for:
1) O157 and H7 antigen using the RIM latex agglutination test
2) Biochemical confirmation using VITEK5
3) Shiga toxin confirmation using EHEC Kit.
Day 4/Day 5

O157 (+) (+) (-)

H7 (+/-) O157 (+) O157 (-)
VITEK (+) H7 (+/-) H7 (-)
Shiga toxin (-) VITEK (+) VITEK (-)
Requires additional Shiga toxin (+) Shiga toxin (-)
testing by OSEL6 Report as CONFIRMED POSITIVE Report as NEGATIVE

CONFIRMED POSITIVE ISOLATES

FSIS Laboratories: Pick isolates to nutrient Pick isolates to cryobeads for storage.
agar deep for PFGE testing at OSEL6

1 Modified Tryptone Soya Broth

2 Modified Rainbow Agar
3 E.coli O157:H7 Latex Test
4 Sheep Blood Agar
5 Automated Microbiology Test System
6 Outbreak Section of the Eastern Laboratory, USDA-FSIS

MLG 5 Appendix 1.02

Page 1 of 1
Effective: 06/29/14 Issuing Authority: Director, Laboratory Quality Assurance Staff (LQAS)