Identification of E. Coli O157 by DYNABEADS® Anti- E coli O157

Laboratory Procedure MFLP-90
July 2006

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1. Application

This method is applicable to the isolation of viable Escherichia coli O157 in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. When product-based compliance action is anticipated, and where stipulated HPFB methodology or MFLP-80 (8.1) is used for final confirmation of isolates (Section 7.). This revised method replaces MFLP-90, dated April 1997.

2. Principle

The Dynabeads® anti- E coli O157 test offers a rapid culture technique based on the selective enrichment of E coli O157 directly from pre-enriched samples using the technology of ImmunoMagnetic Separation (IMS). The basis of IMS is a uniform, superparamagnetic, polystyrene microsphere called Dynabeads®. In this assay, Dynabeads® are coated with absorbed and affinity purified anti - E coli O157 antibodies. The beads are incubated with 1ml of pre-enriched sample. During this incubation, E coli O157 present in the sample will bind to the surface of the bead. The bead-bacteria complexes are separated from the sample using a magnetic particle concentrator (Dynal MPC-S®). The bead-bacteria complexes are then plated onto selective culture media. After incubation, the plates are examined for colonies which exhibit characteristics typical for the growth of E coli O157.

3. Definition of terms

See Appendix A of Volume 3.

4. Collection of samples

See Appendix B of Volume 3.

Dynabeads® and Dynal MPC® are trademarks of Dynal A.S Oslo, Norway. Dynal products are available through Invitrogen Canada Inc., 2270 Industrial St., Burlington, ON. Telephone (800) 263-6236, FAX (800) 387-1007.

5. Materials and special equipment

The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of individual media.

1. Dynabeads® anti - E coli O157, available in two formats; sufficient reagent for either 50 or 250 tests.
2. The Dynal MPC®-S unit is required to perform the test.
3. Enrichment broth, isolation media and supplements (see MFLP-80)
4. A sample mixer capable of tilt, swirl or rotational mixing is also required for this procedure. The Dynal Sample Mixer is available from Dynal, Inc., however, many standard laboratory mixers can be adapted for this protocol.
5. PBS-Tween (Available from Sigma Product # P3563)
6. Micropipette (10 - 100 µL)
7. 1 mL dispenser pipette
8. 1.5 mL microcentrifuge tubes
9. Sterile swabs
10. BeadRetriever® automated immunomagnetic separation instrument (optional)

6. Procedure

6.1 Handling of Sample Units

Refer to MFLP

6.2 Preparation for Analysis

Refer to MFLP-80 (or other compendial methods for E coli O157, some of which have different enrichment protocols).

6.3 Preparation of Sample

Refer to MFLP-80. In addition to the protocol in MFLP-80, the enriched sample may be incubated for shorter periods of time (e.g. 6-18 h) if the screening method allows for this. Follow the specific instructions provided in the screening methods, for types of commodities that can be tested with shorter incubation times than are recommended in MFLP-80.

6.4 Automated IMS

Users must aseptically dispense all reagents and samples into the tubes sequentially after the strips of tubes are fitted into the rack.

Place one disposable sample 5-tube strip into a BeadRetriever rack for each sample to be processed and using aseptic technique, dispense reagents into each tube. The tab on the tube strip may be used for labelling samples.

6.4.1 Aseptically add 10 µL of properly mixed Dynabeads® anti- E coli O157 into two sample tubes 1 and 2.

6.4.2 Aseptically add 500 µL of wash buffer to the sample tubes 1 and 2.

6.4.3 Aseptically add 1 mL of wash buffer to tubes 3 and 4 within the strip.

6.4.4 Aseptically add 100 µL of wash buffer to the 5th tube.

6.4.5 Remove the desired tube from rack A and place in rack B (one meter away, to help reduce possible cross contamination). Add 500 µL of a test sample to tubes 1 and 2 and transfer the inoculated tube to rack A. Repeat for the remaining samples.

6.4.6 Aseptically inset the sterile protective tip combs into the instrument.

6.4.7 Insert the rack with filled tubes into the instrument to lock it in place.

6.4.8 Check that everything is properly aligned and close the instrument door.

6.4.9 Select the EPEC/VTEC program sequence by scrolling with the arrow key and press the START button.

6.4.10 While the instrument is in operation, the door must be kept closed. Each processing step and the total time remaining can be followed on the LC display.

6.4.11 At the end of the program run, remove the tube's rack from the instrument and plate the bead-bacteria complexes from the 5^th tube as in 6.6.

6.4.12 Remove the tip combs and discard into a biohazard waste container together with the tube strips.

6.5 Manual IMS

To avoid cross contamination and for safety reason, it is recommended that immunomagnetic separation could be performed using the BeadRetriever®. In the absence of the BeadRetriever®, strict adherence to good laboratory practice and the following instructions are a prerequisite to obtaining valid results.

6.5.1 Remove the magnetic plate and load the necessary number of 1.5 mL microcentrifuge tubes into the Dynal® MPC-S.

6.5.2 Resuspend Dynabeads® anti- E coli O157 until the pellet in the bottom disappears by using a vortex machine. Pipette 20 µL of Dynabeads® anti- E coli O157 and dispense into each tube.

6.5.3 Add 1 mL of the pre-enriched sample aliquot from section 6.3 and close the tube. Change to a new pipette for each new sample.

6.5.4 Invert the Dynal MPC-S® rack a few times. Incubate at room temperature for 10 minutes with gentle continuous agitation to prevent the beads from settling.

6.5.5 Insert the magnetic plate into the Dynal MPC-S®. Invert the rack several times to concentrate the beads into a pellet on the side of the tube. Allow 3 minutes for proper recovery.

6.5.6 Open the tubes cap using a tube opener and carefully aspirate and discard the sample supernatant as well as the remaining liquid in the tube's cap.

6.5.7 Remove the magnetic plate from the Dynal MPC-S®.

6.5.8 Add 1 mL of wash buffer (PBS-Tween). Do not touch the tube with the pipette since this can cross-contaminate the samples as well as the wash buffer. Close the cap and invert the Dynal MPC-S® a few times to resuspend the beads.

6.5.9 Repeat steps 6.5.5-6.5.8

6.5.10 Repeat step 6.5.5-6.5.7

6.5.11 Resuspend the Dynabeads® bacteria complex in 100 µL wash buffer (PBS tween). Mix briefly using a vortex mixer.

6.6 Isolation of E coli O157:H7

6.6.1 After Immunomagnetic separation, transfer 50 µL or one-half of the resuspended beads onto the media found in MFLP-80.

6.6.2 Spread the bead-bacteria complexes over one half of the plate with a sterile swab. This ensures the break-up of the bead-bacteria complexes. Dilute further by streaking with a loop. Always carry the loop back into the previously streaked quadrant several times to ensure that the beads reach a fresh unstreaked quadrant.

6.6.3 Incubate plates as suggested in MFLP-80.

7. Confirmation

Colonies exhibiting characteristic E coli O157 morphology on selective agar should be biochemically and serologically confirmed following the instructions in MFLP-80 (MFLP-80 indicates the number of colonies to screen for confirmation).

8. References

8.1 Warburton, D., and D. Christensen. 2006. Isolation of E coli O157 in Foods (MFLP-80). Volume 3, Compendium of Analytical Methods, Health Canada, Ottawa. Published on the Food Directorate's (Health Canada) website at: http://www.sc-hc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php.

8.2 Invitrogen. March 2005. BeadRetrieve User's Manual.
 http://www.invitrogen.com/content/sfs/manuals/1189_BeadRetriever_Manual.pdf (PDF Version - 3.92 K)