Cat. no. Rev. no.

710.03 710.04 010 009

Wash Buffer (PBS Tween): 0.15M NaCl, 0.01M Sodium-Phosphate buffer, pH 7.4, with 0.05 % Tween-20. (Autoclavable at 121C for 15 minutes) Sorbitol MacConkey agar

7. Aseptically insert the sterile protective tip combs into the instrument. 8. Insert the rack with filled tubes into the instrument to lock it in place. 9. Check that everything is properly aligned and close the instrument door. 10. Select the EPEC/VTEC program sequence by scrolling with the arrow key and press the START botton. 11. 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. 12. At the end of the program run, remove the tubes rack from the instrument and plate one half of the bead-bacteria complexes from the 5th tube onto each of the appropriate plating media as recommended below (see Section Detection & Confirmation of E.coli O157). 13. Remove the tip combs and discard into a biohazard waste container together with the tube strips. 2.2.2 Immunomagnetic Separation - Manual Method NOTE: To avoid cross-contamination and for safety reasons, it is strongly recommended that immunomagnetic separation should 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. 1. Remove the magnetic plate and load the necessary number of 1.5 ml microcentrifuge tubes into the Dynal MPC-S . 2. Resuspend Dynabeads anti-E.coli O157 until the pellet in the bottom disappears by vortexing. Pipette 20 l of Dynabeads anti-E.coli 0157 and dispense into each tube. 3. Add 1 ml of the pre-enriched sample aliquot and close the tube. Change to a new pipette for each new sample. 4. Invert the Dynal MPC-S rack several times. Incubate at room temperature for 10 minutes with gentle continuous agitation to prevent the beads from settling (e.g. in a Dynal MX4 sample mixer). 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 the tube to stand for 3 minutes for maximum recovery of Dynabeads anti-E.coli O157. NOTE: The magnetic plate for the Dynal MPC-S has two positions for insertion. The vertical position is intended for use with round-bottomed or conical microcentrifuge tubes for larger volume applications (0.5 - 2 ml). The tilted position is for conical microcentrifuge tubes only and is better for lower volume applications (0.01 - 0.5 ml). 6. Open the tube cap using the tube opener provided and carefully aspirate and discard the sample supernatant as well as any remaining liquid in the tube's cap. (See Section 2.4 Factors which affecting the products performance). 7. Remove the magnetic plate from the Dynal MPC-S. 8. Add 1 ml of wash buffer using a different disposable pipette or tip for each sample to prevent cross contamination between samples as well as the wash buffer. Close the cap and invert the Dynal MPC-S several times to resuspend the beads. 9. Repeat steps 5 - 8. 10. Repeat steps 5 - 7. 11. Resuspend the Dynabeads-bacteria complex in 100 l of wash buffer using a different disposable pipette or tip for each sample. Mix briefly by vortexing.

All reagents should be of analytical grade.

Dynabeads anti-E.coli O157

For rapid, selective enrichment of Escherichia coli O157 For research use only INDEX 1 PRODUCT DESCRIPTION 1.1 1.2 1.3 1.4 1.5 1.6 Intended Use Intended User Sample Matrix Principle Interpretation Criteria Description of Materials 1.5 Interpretation Criteria The test is based on plating the concentrated bead-bacteria complexes onto internationally accepted E.coli O157 culture media, such as Cefixime Tellurite Sorbitol MacConkey agar (CT-SMAC) and CHROMagar O157. Interpretation of presumptive results depends on the skill of the user to correctly identify and differentiate the isolated colonies based on typical E.coli O157 morphology. Suspect colonies must be confirmed by standard biochemical and serological test methods. 1.6 Description of Materials Dynabeads anti-E.coli O157 are uniform, superparamagnetic, polystyrene microscopic beads with adsorbed and affinity purified antibodies against E.coli O157 covalently bound to the surface. The beads are supplied in a suspension of phosphate buffered saline (PBS) pH 7.4 with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN33). Sufficient Dynabeads anti-E.coli O157 are provided to perform either 50 or 250 tests (Product Numbers 710.03 & 710.04 respectively). Additional Materials & Equipment Required & Supplied By Invitrogen Dynal For performing AIMS: Component A bench top instrument for performing automated IMS BeadRetriever Tubes and Tips Disposable sample tube strips and tip combs to protect the magnetic probes for use in the BeadRetriever For performing manual IMS: Component Magnetic particle concentrator (Dynal MPC)-S Dynal-MX4 Dynal Sample Mixer (US only) Other materials: Component CT-supplement CHROMagar O157 Prod No. Pack Size 740.01 740.02 1000 tests 4 x 250 ml Prod No. Pack Size 120.20D 1 Unit 159.51 240 Tests Prod No. Pack Size 1 Unit BeadRetriever instrument 159.50

NOTE: There are alternative pre-enrichment broths that have been used successfully prior to IMS. Invitrogen Dynals recommendation for choice of pre-enrichment broths is based on in-house testing and practicality with respect to international regulations. 2 PROTOCOLS 2.1 Sample Preparation Food samples Weigh 25 g of food sample and place into a filter homogeniser bag. Add 225 ml of the enrichment medium (e.g. BPW, TSB, or BGBB) and homogenise. Incubate for 6 -18 h at 37C or 41.5C. Mix the pre-enriched sample thoroughly by homogenising once more. Human stools, bovine faeces and environmental swab samples For human and animal stool samples, prepare a 10% suspension in physiological saline and transfer 1 ml into 10 ml of a suitable enrichment broth. Human rectal swab and environmental swabs samples should be transferred into 10 ml of a suitable enrichment broth. Incubate as described above for food samples. Water samples Filter 1 litre of water according to standard local procedures. Use a flat-ended forceps to remove the filter and transfer directly into a wide-mouthed bottle. Add 90 ml of BPW or TSB to the contents of the bottle and shake vigorously to dislodge bacteria from the surface of the membrane. Incubate for 6-24 h at 37C or 41.5C. The use of a filter aid is recommended for samples that are too turbid for membrane filtration. 2.2 Performing Immunomagnetic Separation 2.2.1 Automated Immunomagnetic Separation (AIMS) Using Dynabeads Anti-E.coli O157 & BeadRetriever NOTE: Please carefully read the instrument operating instructions of the Bead Retriever before use. Place one disposable sample tube strip into a BeadRetriever sample 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. 1. Resuspend Dynabeads anti-E.coli O157 until the pellet in the bottom disappears by vortexing. 2. Aseptically add 10 l of properly mixed Dynabeads anti-E.coli O157 into the two sample tubes 1 and 2. 3. Aseptically add 500 l of wash buffer to sample tubes 1 and 2. 4. Aseptically add 1 ml of wash buffer to tubes 3 and 4 within the strip. 5. Aseptically add 100 l of wash buffer to the 5th tube. 6. Remove the desired tube from the sample rack and place in a second sample rack one metre away. Add 500 l of a test sample to tubes 1 and 2 and transfer the inoculated tube back to the first sample rack. Repeat for the remaining samples.

2 PROTOCOLS 2.1 Sample Preparation 2.2 Performing Immunomagnetic Separation 2.2.1 Automated Immunomagnetic Separation (Aims) Using Dynabeads Anti-E.coli O157 & BeadRetriever 2.2.2 Immunomagnetic Separation Manual Method 2.3 Specificity And Sensitivity 2.4 Factors Which Affect The Products Performance 2.5 Precautions/Limitations 3 GENERAL INFORMATION 3.1 3.2 3.3 3.4 3.5 3.6 Storage/Stability Technical Service Warnings and Limitations Trademarks Limited Use Label License Warranty

4 REFERENCES 1 PRODUCT DESCRIPTION 1.1 Intended Use For the rapid selective separation of E.coli O157:H7 from food, water or environmental samples. This process can be automated using a BeadRetriever bench top instrument or performed using a manual method. 1.2 Intended User Any laboratory skilled in using conventional microbiological techniques, equipped and/or certified to do pathogen testing on food, feed and environmental samples, may use Dynabeads anti-E.coli O157. The user must be skilled in using conventional microbiological techniques and in interpreting results. 1.3 Sample Matrix Any food, water, feed or environmental samples that has been pre-enriched for 6-18 hours in Buffered Peptone Water (BPW), Tryptone Soya Broth (TSB) or Brilliant-Green Bile Broth (BGBB) is suitable for IMS with Dynabeads anti-E.coli O157. 1.4 Principle Dynabeads anti-E.coli O157 are designed for rapid, selective concentration of E.coli O157 directly from a pre-enriched sample aliquot using immunomagnetic separation (IMS). Dynabeads anti-E.coli O157 reacts with all E.coli O157 strains including pathogenic and non-pathogenic, sorbitol fermenting and non-sorbitol fermenting isolates. Dynabeads anti-E.coli O157 are simply incubated with an aliquot of the pre-enriched sample and the antibodies coated onto the beads will specifically bind the target bacteria. The bead-bacteria complexes are subsequently separated by applying a magnetic field. The whole IMS process can be automated using a BeadRetriever instrument or performed manually.

159.10 947.01

1 Unit 1 Unit

Additional Materials & Equipment Needed & Not Supplied By Invitrogen Dynal Micropipette (10 - 100 l) 1 ml dispenser pipette Pre-enrichment broths such as BPW, TSB, BGBB or other pre-enrichment broths Stomacher and stomacher bag with filter Test tubes, glassware, loops, swabs and pipettes

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12. The enriched bacteria on the beads are now ready for use in the detection step (See Section Detection & Confirmation of E.coli O157 below). Detection & Confirmation of E.coli O157 After IMS, transfer the resuspended beads onto each internationally accepted E.coli O157 culture media plates. It is recommended that two different culture media are used to increase the chances of detecting suspect colonies that have distinct differential features on each media Invitrogen Dynal recommends Sorbitol MacConkey (SMAC) media supplemented with CT-supplement and CHROMagar O157. 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. Incubate the plates at 35-37C for 18-24 h. Read the plates for suspect E.coli O157 colourless colonies on CT-SMAC and pink-mauve coloured colonies on CHROMagar O157. The choice of plating media has been based on some distinct characteristics of E.coli 0157:H7. It is the only E.coli among clinical isolates which does not ferment sorbitol within 24 h and which is glucuronidase negative. The organisms are resistant to potassium tellurite and cefixime (See Section 4.REFERENCES). Presumptive E.coli O157 colonies must be confirmed by standard biochemical and serological testing. 2.3 Specificity And Sensitivity The protocol for use with Dynabeads anti-E.coli O157 will determine the presence or absence of one viable E.coli O157 in the sample size described if this one cell is able to replicate and is not competed out by resident background flora. Dynabeads anti-E.coli O157 will bind both motile and non-motile strains of E.coli O157. The binding is independent of the ability to produce either Shiga toxins 1 or 2, or both. Antigenically similar organisms, for example Escherichia hermanii, Salmonella O group N, or Proteus spp., can crossreact and bind to a limited extent. In addition extremely "sticky" organisms like Pseudomonas spp. or Serratia liquifaciens could bind non-specifically. However the presence of high numbers of competitive background flora in the sample will not affect the binding of E.coli O157 to the beads. In naturally contaminated samples the Dynabeads anti-E.coli O157 protocol in combination with CT-SMAC agar can detect E.coli 0157 from preenriched sample aliquots containing as low as 100 E.coli O157 cells against high numbers of background flora of 106 organisms or more per ml. False Negatives/Positive Rates A false negative rate ranging between 2-10% may be expected using the Dynabeads anti-E.coli O157 protocol depending on the inoculum level, background flora and sample matrix. However in identical samples tested without IMS, this false negative rate is significantly increased and is often more than 25%. Therefore use of the Dynabeads antiE.coli O157 protocol will consistently decrease the sample false negative rate by more than 15%. False positives do not occur since all presumptive colonies must always be verified by suitable identification methods. However the methods depends on the user following good laboratory practices and avoiding cross-contamination of samples. The accuracy of the method is not measurable since IMS is a qualitative and not a quantitative technique. Several bacteria may be bound to the Dynabeads, but only give rise to one colony forming unit on the culture media. The precision is dependent on the extent to which

particles are recovered from different sample matrices. 2.4 Factors Which Affect The Products Performance The IMS procedure should be performed on a bench-top at room temperature between 1525C and all reagents must be at room temperature before use. Ensure that the Dynabeads anti-E.coli O157 are fully dispersed by vigorous vortexing for at least 10 seconds before use. It is important that filtered pipette tips are used to transfer samples into the test tubes for both manual and automated IMS. When performing manual IMS the user must take care not to aspirate and discard the isolated bead-bacteria complexes. The use of vacuum aspirators has been shown to reduce the recovery of bacteria. If Dynabeads-bacteria complexes are accidentally aspirated from the sample tube, immediately dispense back into the tube and dilute with wash buffer, repeat step 5 in Section 2.2.2 Immunomagnetic Separation - Manual Method before aspirating again. Failure to recover the bead-bacteria complexes could result in failure to isolate E.coli O157. If aspiration becomes difficult, leave some of the supernatant in the tube and dilute with washbuffer. In fatty samples, this will break the fat content which causes the Dynabeads to slide down the tube wall. In extremely fatty, viscous or particulate samples, a two-fold sample dilution using the wash buffer must be made prior to IMS to ensure maximum particle recovery. During bead-bacteria complex magnetic capture it is essential that gentle rocking of the Dynal MPCs is continued. This prevents binding of low mass debris, which is magnetic or magnetisable. For automated IMS to avoid cross-contamination of the prepared tubes it is recommended that sample transfer into the tubes is performed in a designated area at least one metre from the prepared tubes.

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at http://www.invitrogen.com. 3.4 Trademarks Dynal, Dynabeads, BeadRetriever and Dynal MPC are either registered trademarks or trademarks of Invitrogen Dynal AS, Oslo, Norway. Any registration or trademark symbols used herein denote the registration status of trademarks in the United States. Trademarks may or may not be registered in other countries. CHOMagar O157 is a registered trademark of Dr A. Rambach, Paris, France. Intellectual Property Disclaimer Invitrogen Dynal will not be responsible for violations or patent infringements that may occur with the use of our products. 3.5 Limited Use Label License No. 5: Invitrogen Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500. Email: outlicensing@invitrogen.com 3.6 Warranty The products are warranted to the original purchaser only to conform to the quantity and contents stated on the vial and outer labels for the duration of the stated shelf life. Invitrogen Dynal's obligation and the purchaser's exclusive remedy under

this warranty is limited either to replacement, at Invitrogen Dynal's expense, of any products which shall be defective in manufacture, and which shall be returned to Invitrogen Dynal, transportation prepaid, or at Invitrogen Dynal's option, refund of the purchase price. Claims for merchandise damaged in transit must be submitted to the carrier. This warranty shall not apply to any products which shall have been altered outside Invitrogen Dynal, nor shall it apply to any products which have been subjected to misuse or mishandling. ALL OTHER WARRANTIES, EXPRESSED, IMPLIED OR STATUTORY, ARE HEREBY SPECIFICALLY EXCLUDED, INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Invitrogen Dynal's maximum liability is limited in all events to the price of the products sold by Invitrogen Dynal. IN NO EVENT SHALL INVITROGEN DYNAL BE LIABLE FOR ANY SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES. Some states do not allow limits on warranties, or on remedies for breach in certain transactions. In such states, the limits set forth above may not apply. 4 REFERENCES ISO 16654:2001 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157 Bacteriological Analytical Manual Online Chapter 4A Diarrheagenic Escherichia coli U.S. Food & Drug Administration, Center for Food Safety & Applied Nutrition 2002 Fegan N, Higgs G, Vanderlinde P, Desmarchelier P. Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation. Lett Appl Microbiol. 2004;38(1):56-9 Fegan N, Higgs G, Vanderlinde P, Desmarchelier P. Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation. Lett Appl Microbiol. 2004;38(1):56-9 Bennett AR, MacPhee S, Betts RP The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures. Lett Appl Microbiol. 1996; Mar22(3):237-43 Chapman PA, Wright DJ, Siddons CA A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces.J Med Microbiol. 1994 Jun;40(6):424-7 Wright DJ, Chapman PA, Siddons CA Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol Infect. 1994 Aug;113(1):31-9 Zadik PM, Chapman PA, Siddons CA 1993 Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Micro. 39: 155-158 Okrend, AJG, Rose BE, Lattuada CP 1990 Use of 5-Bromo-4-Chloro-3-Indolyl-J-D-Glucuronide in MacConkey Sorbitol Agar to Aid in the Isolation of Escherichia coli O157:H7 from Ground Beef J. Food Prot. 53: 941-943

2.5 Precautions/Limitations In order to obtain a homogeneous dispersion of beads in suspension, resuspend Dynabeads antiE.coli O157 by using a vortex until the pellet in the bottom disappears before use. Precautions should be taken to prevent bacterial contamination of opened vials. All material that is used and contaminated should be autoclaved and properly disposed of according to local regulations. 3 GENERAL INFORMATION Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003. 3.1 Storage/Stability This product is stable until the expiry date stated on the label when stored unopened at 2-8C. Store opened vials at 2-8C and avoid bacterial contamination. Keep Dynabeads in liquid suspension during storage and all handling steps, as drying will result in reduced performance. 3.2 Technical Service Contact details for your local Invitrogen technical support can be found at www.invitrogen.com 3.3 Warning And Limitations This kit is for research use only. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic. Avoid pipetting by mouth!

Invitrogen Dynal is a part of the Invitrogen Group. Contact details for your local Invitrogen sales office/technical support can be found at http://www.invitrogen.com/contact Copyright 2007 Invitrogen Dynal AS, Oslo, Norway. All rights reserved.
SPEC-06020 Revised: 06.2007 Printed: 06.2007

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