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Supplement 2 to Method MFLP-30 The Use of the BAX E. COLI O157:H7 MP Assay
Supplement 2 to MFLP-30
November 2006
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(PDF Version - 21 K)Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204A1
Ottawa, Ontario K1A 0L2
Email: Don_Warburton@hc-sc.gc.ca
1. Application
The following information is offered as a supplement to the method MFLP-30, dated March 2003, and should be used with this method. The purpose of this supplement is to provide additional information (such as source(s) of materials, critical steps, confirmation steps, or helpful hints etc.).
The Bax E. coli O157:H7 MP Assay may be substituted for the kit described in MFLP-30.
Using the enrichment broth from MFLP-80 (mTSB-n), HC and CFIA labs have validated the use of the BAX System E. coli O157:H7 MP kit for dairy, meat, fruit and vegetables, and dry products.
It is the responsibility of the Laboratory Supervisor to properly validate the kit and media for all other types of samples.
6. Materials and Special Equipment
- BAX System E. coli O157:H7 MP media (for beef only)
- Modified Tryptic Soy Broth with Novobiocin (m-TSB-n) (see Supplement 1 to MFLP-30)
- Buffered Peptone Water (BPW) for dry foods and samples
- BAX System E. coli O157:H7 MP test kits.
7. Procedure
7.1 Use the following information in conjunction with MFLP-30. Prepare the required volume of either the BAX system MP media for E. coli O157:H7 according to instruction on the container label or or mTSB-n following instructions in Appendix G.
7.1.1 Prepare a 1:10 dilution of the food by aseptically adding 25 g or mL (or other suitable analytical unit) into 225 mL (or amount necessary to maintain the 1:10 ratio) of the enrichment broth, specified below. Stomach or blend.
7.2 Using BAX System E. coli O157:H7 MP media:
7.2.2 Beef - using the BAX system MP media enrichment: Add raw beef to pre warmed BAX system MP media broth. Incubate at 42ºC for 8 - 24 hours.
7.3 Using mTSB-n (MFLP-80):
7.3.1 Dairy, Meat, and Fruit and Vegetables - Add pre warmed mTSB-n and incubate at 42ºC for 18 -24 hours.
7.3.2 Dry Foods and Other Dry Samples (e.g., environmental samples and animal feed) - Add sufficient BPW to the sample to moisten, equillabrate 1 h at 35ºC. Add pre warmed mTSB-n and incubate at 42ºC for 18 - 24 hours.
After incubation at 42ºC for 18 - 24 hours, remove aliquots to proceed with analysis via Bax. Re-incubate samples if necessary while the BAX test is proceeding, to ensure that the required 22 to 24 hours of incubation have been completed if confirmation by MFLP-80 is required.
7.4 Bax Equipment Preparation:
Refer to MFLP-30.
7.5 Lysis:
7.5.1 Using BAX System E. coli O157:H7 MP media: For beef products incubated in the BAX System E. coli O157:H7 MP media, transfer 20 μL to the corresponding lysis tube.
7.5.2 Using mTSB-n (MFLP-80): Transfer 5 μL from the samples that were incubated in mTSB-n to the corresponding lysis tube.
7.5.3 Continue with MFLP-30.
7.6 Confirmation:
Follow MFLP-80 for selective isolation and confirmation steps.
