United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 1 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

1. Purpose

This Standard Operating Procedure (SOP) will be used after obtaining a positive PCR 

result for Escherichia coli (E. coli) O157:H7 (SOPs MDP MTH-11 and/or MDP 

MTH-12). This document provides standard procedures for capturing E. coli O157 

cells by immunomagnetic separation, subculturing to various selective agar media, 

and identifying isolates by VITEK®. Isolates can be further identified by serotyping. 

2. Scope

This SOP shall be followed by all laboratories conducting microbiological studies for

MDP, including support laboratories conducting non-routine activities that may 

impact the program. This SOP represents minimum MDP requirements and is 

presented as a general guideline. Each laboratory shall have written procedures that 

provide specific details concerning how the procedure has been implemented in that 

laboratory. IMS is used as one of the methods in the isolation of E. coli O157. 

3. Principle

The immunomagnetic separation (IMS) method offers a means to concentrate target 

bacteria from mixed cultures by physical separation based on an antigen-antibody 

reaction. Selective capture (concentration) of bacterial cells is achieved by antibodies, 

specific to the cell surface antigens of the target strain, immobilized on magnetic 

beads. After washing the beads to remove non-target organisms, the magnetized 

beads coated with target bacteria are recovered and processed for isolation using 

various selective agar media and identification by cultural methods and serotyping. 

4. Outline of Procedures

Equipment and Materials.... .................. 6.1

Media and Reagents............................ 6.2

Controls.......................................... 6.3

Safety............................................. 6.4

Isolation of E. coli O157...................... 6.5

Identification of E. coli O157:H7........... 6.6

Reporting........................................ 6.7
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 2 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

5. References

 BAM Online, 8

th edition. 2001. Chapter 4. Section: Diarrheagenic Escherichia 

coli. Isolation and confirmation of E. coli O157:H7. 

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalA

nalyticalManualBAM/UCM070080 (last accessed 03/11) 

 Enrichment of E. coli O157, Product information for Dynabeads® anti-E.coli 0157 

http://www.invitrogen.com/site/us/en/home/brands/Dynal.html? (last accessed 

03/11)

 SOP MDP-DATA-01, Microbiological Record Keeping and Results Reporting

 SOP MDP-SHIP-03, Procedures for Packaging, Shipping, and Archiving 

Microbiological Cultures

 SOP MDP-QA-03, Quality Assurance (QA) Controls

 USDA/FSIS Microbiology Laboratory Guidebook Chapter 5.05, Effective date:

10/1/2010. Detection, isolation, and identification of Escherichia coli O157:H7 

and O157 from meat products. http://www.fsis.usda.gov/PDF/MLG_5_05.pdf 

(last accessed 03/11)

6. Specific Procedures

6.1 Equipment and Materials

o Additional materials needed to perform procedure as listed in the 

product‟s user instructions or suggested by the manufacturer

o Laboratories that have Pathatrix may use it in place of the Dynal system. 

Additional materials needed to perform procedure as listed in the Pathatrix 

(Matrix MicroSciences) and E. coli O157 test kit product instruction or 

suggested by the manufacturer

o VITEK®(bioMerieux, Inc.) System and Users Manual

o VITEK Cards: GNI+ and GN cards, bioMerieux

o Incubator 35 ± 2oC

o Incubator 42 ± 2oC
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 3 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

 6.2 Media and Reagents

o Dynabeads®anti-E. coli O157 (Dynal Biotech), Invitrogen

o Optional: Ultra E. coli O157 test kit, Matrix Microsciences

o Wash buffer (PBS Tween): 0.15M NaCl, 0.01M Sodium-Phosphate 

buffer, pH 7.4, with 0.05% Tween-20; autoclaved at 121oC for 15 minutes

o CHROMagarTM O157, DRG International, R&F®, (or demonstrated 

equivalent) with supplements (e.g. tellurite, cefixime, cefsulodin etc.)

o TCSMAC (Tellurite-Cefixime Sorbitol MacConkey Agar) plates

o TP (Tryptone Phosphate) Broth

o Modified Buffered Peptone Water plus pyruvate (mBPWp) with ACV

o TSAYE (Trypticase Soy Agar with Yeast Extract) 

o BA (Blood Agar) plates

o Commercially available agglutination test kits for somatic O and flagellar 

H antisera for E. coli O157:H7 (Lenexa or Remel KS or products with 

demonstrated equivalent)

o Reagents for MUG based test (e.g. ColiComplete Discs, (BioControl Co. 

Bellevue, WA) or equivalent)

o Kovac‟s reagent for Indole test (or equivalent such as BBL Dryslide 

Indole)

 6.3 Controls: (Specific strains are listed in SOP MDP-QA-03, Attachment 1).

6.3.1 For IMS, at a minimum, carry positive produce control (spiked with MDP 

004) from SOP MDP-MTH-11 through this entire procedure. Refer to SOP MDPLABOP-02 for control setup. 

NOTE: Alternatively, it is acceptable for laboratories to set up appropriate media controls 

(positive, negative, and uninoculated) from pure cultures without using the cultural 

controls from the screening methods. Each laboratory shall have written procedures for 

ensuring the appropriate controls are used for this procedure. 

6.3.2 If any of the controls fail to yield a satisfactory result refer to SOP MDPQA-03.

 6.4 Safety - E. coli O157:H7 is a human pathogen with a low infectious dose. 

Laboratory personnel should utilize Biosafety Level II (BSL-2) practices for 

microbiological manipulations of known and potential pathogens. A BSL-2 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 4 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

laminar flow biosafety cabinet is recommended for activities with potential for 

producing aerosols of pathogens. Material Safety Data Sheets (MSDS) should be 

obtained from manufacturers for media, chemicals and reagents used in the 

analysis and personnel who will handle the materials should know the location of 

and have ready access to the MSDS sheets for reference.

 6.5 Isolation

6.5.1 Transfer 10 ml of the pooled PCR-positive UPB sample (identified positive 

for E. coli O157:H7 by SOPs MDP MTH-11 and MTH-12) to one 90ml TP broth 

and 10ml pooled PCR-positive UPB to one 90ml of mBPWp w/ACV and 

incubate them at 44 ± 2

oC and 42 ± 2°C, respectively, for 18-24 hours. Transfer 

10ml of the individual (sprouts) PCR-positive UPB sample to 90ml of TP broth 

and 10ml individual PCR-positive UPB to 90ml mBPWp w/ACV and incubate at 

44 ± 2°C and 42 ± 2°C, respectively, for 18-24 hours.

6.5.2 Extract DNA and repeat BAX® O157:H7 Rt-PCR on TP and mBPWp 

w/ACV enrichments. If negative results are obtained, return to the UPB enriched 

positive samples, following procedures in section 6.5.6, this SOP.

6.5.4 Streak the PCR-positive TP and/or mBPWp w/ACV enrichments (pooled 

and/or individual samples) onto selective agar plates, CHROMagarTM O157 and 

TCSMAC, for isolation. Incubate one set of selective agar plates at 35 ± 2oC for 

18-24 hours. Incubate a second set of agar plates at 42 ± 2oC for 18-24 hours.

6.5.5 Streak positive and negative control cultures on selective agar plates and 

incubate the plates at 35 ± 2oC for 18-24 hours.

Note: Use of several selective agar plates, both in type and number, will improve the 

chance of finding the target isolates on plates. Use two types of selective agar plates, 

CHROMagar O157 and TCSMAC for screening at two different temperatures. BAX®

E. coli O157:H7 realtime PCR on TP-enriched cultures can be performed to verify the 

presence of E. coli O157:H7.

6.5.6 Perform IMS on all BAX®-positive pooled and individual enrichments 

(UPB, TP, and/or mBPWp w/ACV) following the manufacturer‟s instructions or 

validated IMS procedure. Include positive produce control sample. 

6.5.7 Dispense the concentrated samples to several plates of CHROMagarTM O157 

and TCSMAC and streak for isolation following Dynal/Pathatrix IMS 

manufacturer‟s instructions. Incubate the plates at 35 ± 2oC and the duplicate 

plates at 42 ± 2oC for 18-24 hours. 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 5 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

 6.5.8 Observe all control and sample plates for typical E. coli O157 colonies.

6.5.9. Pick a minimum of 10 typical colonies (if available) from any of the 

selective agar plates used for isolation. Select presumptive positive colonies and 

perform latex agglutination for O antigen. Select up to a total of five isolates and 

streak „O‟ positive colonies to BA and/or TSAYE. Include the positive and 

negative controls. (Aseptically add a ColiComplete disc to the primary streak on 

the TSAYE agar plates. Incubate TSAYE Agar plates for 18-24 hours at 35±2o 

C.) 

6.5.10 Check for MUG reaction using UV light for fluorescence. (E. coli

O157:H7 is a MUG negative organism.)

6.5.11 Check Indole test.

6.5.12 Perform latex agglutination test for O and H antigens on typical colonies. 

Include positive and negative controls and verify GFP absence for positive 

samples.

Note: Hold CHROMagar and TCSMAC plates at refrigeration temperature until 

results are confirmed.

 

6.6 Identification of E. coli O157:H7

6.6.1 Select 3-5 MUG negative, Indole positive, O157 antigen positive typical 

colonies from BA and identify using VITEK®or another official standard method 

of identification. 

6.6.2 Perform real-time PCR to confirm the presence or absence of stx-1 and stx-2

genes in the isolates. Refer to SOP MDP MTH-11. 

Typical colony characteristics of E. coli O157:H7

Medium/Test Colony Characteristics Phenotype

CHROMagarTM O157 Mauve Proprietary physiological test for O157

TCSMACTM Colorless with gray center Sorbitol negative

Indole Test Positive
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 6 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

6.6.3 Select one typical colony that has been confirmed by VITEK®, serotyping, 

and confirmatory Rt-PCR as E. coli O157:H7 for archiving and shipping. 

6.7 Reporting and Shipping

6.7.1 A final positive result is defined as a culture that is biochemically identified 

as E. coli O157:H7 and serologically confirmed as E. coli O157:H7. 

6.7.2 IMMEDIATELY following completion of biochemical and serological 

tests, report confirmed results to MP per SOP MDP-DATA-01 on Attachment 1, 

Results Notification Form, and prepare (3) isolate slants for shipment to MDP 

repository for antimicrobial susceptibility testing, and PFGE (if not done by the 

testing laboratory).

 6.7.3 Refer to SOP MDP-SHIP–03 for preparation of cultures for shipment.

Disclaimer: Reference to brand names (kits, equipment, media, reagents, etc.) 

does not constitute endorsement by this agency
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 7 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

E. coli O157:H7 Isolation

BAX E. coli O157:H7 positive 

UPB/TP/mBPWp w/ACV 

pooled/individual enrichments

Rt-PCR of isolate(s)

3-5 typical colonies on 

VITEK

Select one isolate 

for shipping

IMS

Streak on TCSMAC and 

CHROMagarTM O157

Streak on TSAYE/BA 

for single/pure colony 

isolation

Minimum of 10 „O‟ 

positive colonies

Test for latex agglutination, 

MUG, Indole, and GFP
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 8 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

Mary Tijerina 22 June 2011

___________________________________________________________

Revised by: Mary Tijerina Date

Microbiologist, Microbiological Data Program

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x111

Maya Achen 27 June 2011

_____________________________________________________________

Reviewed by: Maya Achen Date

Chairperson, MDP Technical Advisory Group

Ohio Department of Agriculture Consumer Analytical Laboratory

Bldg 3, 8995 E Main Street, Reynoldsburg, OH 43068

(614) 728-2790

Shanker Reddy 6 July 2011

_____________________________________________________________

Reviewed by: Shanker Reddy, PhD Date

Molecular Biologist, Microbiological Data Program

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x135

Diana Haynes 7 July 2011

Approved by: Diana Haynes Date

Deputy Director, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x134

ELECTRONICALLY REPRODUCED SIGNATURES - ORIGINAL SIGNATURE PAGE MAINTAINED BY USDA, AMS, S&T, MONITORING PROGRAMS 

OFFICE
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 9 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

Revision 07 May 2011 Monitoring Programs Division

 Changed word “Dynabeads®” to beads, Section 3.

 Revised “Equipment and Materials”, Section 6.1 by adding words in bold:

 Additional materials needed to perform procedure as listed in a product’s

user instructions or as suggested by the manufacturer.

 Laboratories that have Pathatrix may use it in place of the Dynal 

system.

 Deleted “mEC+n” from SOP

 Revised “Media and Reagents”, Section 6.2 by adding:

o Optional: Ultra E. coli O157 test kit, Matrix Microsciences

o Kovac‟s reagent for Indole test (or equivalent such as BBL Dryslide 

Indole)

o Modified Buffered Peptone Water plus pyruvate (mBPWp) with ACV

 Revised Section 6.3.1 to read as “For IMS, at a minimum, carry positive produce 

control...”

 Revised old Section 6.5 to be more specific by adding new sub-sections: “6.5.1, 

6.5.2, 6.5.3, 6.5.4, 6.5.5” and renumbered old subsections (6.5.2 and 6.5.3) as 

6.5.6 and 6.5.7

 Added “Indole Test” to Table of Typcial colony characteristics of E. coli 

O157:H7.

 Revised and combined old Sections 6.5.5 and 6.5.6 (now 6.5.9) to change 

minimum number of colonies (in bold) “Pick a minimum of 10 typical colonies (if 

available) from any of the selective agar plates....” 

 Added Section 6.5.11, “Check Indole test.”

 Revised old Section 6.5.7 (now 6.5.12) to read as “Perform latex agglutination 

test for O and H antigens on typical colonies. Include positive and negative 

controls and verify GFP absence for positive samples.” 

 Revised Section 6.6.1 to read as “Select 3-5 MUG negative, Indole positive, 

O157 antigen positive typical colonies from....”

 Updated “E.coli O157:H7 Isolation” Flowchart
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 10 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

Revision 06 June 2010 Monitoring Programs Office

 Removed words “L-EMB, MacConkey, and CHROMagarTM E. coli plates” from 

document

 Added the media and reagent: TSAYE and ColiComplete disc

 Expanded the section 6.5 Isolation

 Removed MTH-05 and MTH-07 from reference

 Updated Flowchart

Revision 05 December 2009 Monitoring Programs Office

 Alphabetized and removed numbering from References, Section 5

 Removed numbering from Equipment and Materials, Section 6.1

 Removed numbering from Media and Reagents, Section 6.2

Changed sentence to “Pick a minimum of 5-10 typical colonies (if available) on 

the selective agar plates used for isolation or restreak to CHROMagarTM...”, 

Section 6.5.5

 Changed Section 6.7.2 to read “IMMEDIATELY following completion of 

biochemical and serological tests, report confirmed results to MP per SOP MDPDATA-01 on Atch 01, Results Notification Form, and prepare (3) isolate slants 

for shipment to ODA for antimicrobial susceptibility testing and, if necessary, 

PFGE and serotyping.”.

 Deleted “Report results according to SOP MDP-DATA-01”, Section 6.7.3

Revision 04 March 2009 Monitoring Programs Office

 Added 5.8 to References

 Added “Invitrogen” to 6.2.1.

 Deleted 6.2.3, mEC with Novobiocin

 Added “mEC + n selectively enriched cultures (pooled and individual samples)” 

to 6.5.1.

 Added “form both the pooled and each individual sample (the 3 samples that 

tested BAX positive from the pooled sample)” to 6.5.2.

 Deleted transferring UPBt to mEC + n from section 6.5.

 Added “pooled mEC + n and individual” to 6.5.3.

 Added 6.6.2.

 Revised the flowchart and removed reference to UPBt and selective enrichment in 

mEC + n.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-06 Page 11 of 11

Title: Isolation and Identification of Escherichia coli O157 by Immunomagnetic Separation (IMS) and Cultural 

Methods 

Revision: 07 Replaces: Rv 6 07/01/2010 Effective: 9/1/2011

Revision 03 April 2007 Monitoring Programs Office

 Added mEC+n broth for enriching BAXPCR E. coli O157:H7 positive cultures at 

42oC and 37oC.

 Added incubation of selective agar plates at two different temperatures, 37 and 42oC.

Revision 02 January 2006 Monitoring Programs Office

 Expanded isolation and identification steps

 Introduced serotyping

 Changed the requirement for IMS processing of a positive sample from one 1.0 mL 

sample to three to five 1.0 mL samples.

 Changed IMS to one of the methods in the isolation process

Revision 01 January 2005 Monitoring Programs Office

 Changed controls

 Modified to refer to manufacturer‟s instructions for the IMS protocol