MPNplate™User Manual and MPN Table

The CPI International MPNplate™ is a simple and easy to use device for enumerating microorganisms in a liquid sample. It is a modern approach to the traditional 15 tube serial dilution method and the statistical model of the most probable number (MPN) index, as published in the Standard Methods for the Examination of Water and Wastewater (American Public Health Association, American Water Works Association, and Water Environmental Federation) and accepted worldwide. Replacing the time consuming and labor intensive 15-tube dilution method, the device has five 10 mL wells, five 1 mL wells, and five 0.1 mL wells. A 16th well for collecting the remaining sample allows a full 100 mL sample to be analyzed. A mixture of reagent and sample is poured directly into the wells, filling all 15 wells first and the overflow well after. The device is then sealed with an adhesive film and set up for incubation. Result reading and MPN determination is direct and simple.

Tests using Colitag™ and the MPNplate™ offer a high degree of sensitivity with a detection limit ranging from <1 to 1,600 MPN per 100 mL sample. The Colitag™ MPN plate™ system requires only an incubator, a long-wave UV lamp, a reagent blank or control strains, sealing film, and the hand-held sealing tool. The entire procedure involves only a few steps and can be done in less than one minute per sample.

For Information about validating reagents/test methods using the MPNplate™ or for technical support, please contact CPI International.

E-mail: info@cpiinternational.com Web: www.cpiinternational.com www.colitag.com

USA: CPI International 5580 Skylane Blvd. Santa Rosa, CA 95403 Phone: 800 878 7654

Europe: CPI International Nieuwe Hemweg 7-P 1013 BG Amsterdam The Netherlands Phone: +31 20 638 05 97

The MPNplate™ is distributed by CPI International. The device and its use are covered by U.S. Patent Application No. 61/589,253 and Chinese Patent Applications Nos. 201210115069.X and 20122016668.X.

CPI International MPNplate™ User Manual and MPN Table V2.0 - June 2017 Page 1

Instructions for Using the Colitag™ and MPNplate™I. SAMPLE PREPARATION

MIX POUR SEAL

1. Using aseptic techniques, combine the contents of one packet of Colitag™ medium with a 100 mL liquid sample. Close the container securely and shake well to dissolve. Pre-warm sample in a 44.5°C water bath for 7 to 10 minutes or alternatively 35°C for 20 minutes if reading results at 16-48 hours. The pre-warming step is not necessary if reading results only between 24-48 hours.

2. Remove the MPNplate™ from its packaging and place it on a level counter. Avoid touching the top surface or the inside of the wells.

4. To seal the MPNplate™, take the sealing film provided and remove the backing from one of the tab sides by folding along the perforation. Carefully center and attach the film to the side of the device. The folded perforated edge should line up with the edge of the device. Press to secure the tab of the sealing film in place.

3. Per Standard Method technique for MPN analyses, ensure the sample has been shaken vigorously at least 25 times before dispensing into plate. Aseptically pour the sample/reagent mixture directly into the wells, starting with the large 10 mL wells, and filling all 15 serial wells to the very top. Pour the remaining sample over the top of the plate or into the largest 16th well, taking care to avoid splashing or formation of bubbles. If a well has bubbles or is not full, use a sterile pipet to remove the bubbles or to transfer some liquid from the 16th well to fill it.

5. Remove the middle piece of the film backing taking care not to touch the sterile adhesive surface. Removal of the backing may be facilitated by twisting the edge of the film near the score line or pushing the film towards the plate.

6. Use one hand to firmly position the film straight over the plate and use the sealing tool to seal the film down over the top of the wells. Avoid trapping bubbles in the wells. Any excess sample will be pushed into the channel that leads to largest well.

7. Remove the third portion of film backing and secure the rest of the sealing film to the end of the device. Fold and attach all edges and corners of the film to completely seal the device. Using a back and forth motion, firmly roll the sealing tool over the top of the device to securely seal individual wells.

CPI International MPNplate™ User Manual and MPN Table V2.0 - June 2017 Page 2

A video showing how to use the MPNplate can be viewed at: http://www.colitag.com/mpnplate

II. INCUBATION AND RESULT READING

8. Incubate the device with the sample at 35±0.5°C or according to local regulations for 16-48 hours if pre-warming step was performed, or 24-48 hours if pre-warming step was omitted. Multiple devices can be stacked if desired. Plates should be placed in the incubator such that air can circulate around them to allow for consistent and accurate incubation temperature.

9. Observe the test results, according to the Colitag™ test method (see instructional insert included with product, available from CPI International). Yellow color greater than the blank or non-coliform control indicates a positive test result for total coliforms. If a total coliform positive result is observed, check for fluorescence with a 6-watt 365nm UV light within five inches (12-13cm) of the sample in the dark. Analysts should wear UV protective safety glasses when operating UV lamp. Blue fluorescence indicates a positive result for E. coli. Count the numbers of positive large (10 mL) wells, medium (1 mL) wells, and small (0.1 mL) wells, and also note the result of the 16th well.

10. Use the numbers of positive wells and the MPNplate™ MPN table on page 4 to determine the MPN value of the sample. If the number combination is not found on the MPN table, results can be obtained from the EPA MPN calculator: https://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=309398

11. Discard used MPNplate™ devices properly according to local regulations Do NOT re-use for other samples.

Important Application Notice: User validation for specific application, reagent, analyte, and sample matrix is required. Contact CPI International by calling 1(800) 878-7654 or 1(707) 525-5788 or by sending an e-mail to info@cpiinternational.com for information on validating reagents and methods. CPI International does not support any claims made by other manufacturers regarding performance of unauthorized applications or method/product capability.

CPI International MPNplate™ User Manual and MPN Table V2.0 - June 2017 Page 3

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