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ATP Bioluminescence for Monitoring Drinking Water Quality

ATP Bioluminescence for Monitoring Drinking Water Quality. JiYoung Lee & Carmen Dumas Rolf A. Deininger & Janice Skadsen. School of Public Health University of Michigan August 2005. Scope of Project. Evaluate Rapid Screening method for bacterial numeration

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ATP Bioluminescence for Monitoring Drinking Water Quality

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  1. ATP Bioluminescence for Monitoring Drinking Water Quality JiYoung Lee & Carmen Dumas Rolf A. Deininger & Janice Skadsen School of Public Health University of Michigan August 2005

  2. Scope of Project • Evaluate Rapid Screening method for bacterial numeration • Correlate current methodology HPC with ATP (RLU) analysis. • Variation of instrument performance if any on different water chemistries: Analyzed 6 surface water and 6 ground water plants in Michigan. • Generate a working SOP and training disk for using the Luminos instrument.

  3. Michigan Section Laboratory Practice Committee Special Projects Grant ATP Luminos Michigan Section American Water Works Association

  4. Problems of Current Test Methods • The test results can not be obtained before water is consumed. • Standard Methods: • R2A: most sensitive method (3~7 days)

  5. Ideal Analysis • Rapid • Sensitive • Quantitative • Differentiate between dead and viable • Comparable with current methods • Field applicable • Simple • Cost effective

  6. ATP bioluminescence Luciferin + ATP Oxyluciferin Luciferase Mg2+ O2 hu + PP Luciferyl Adenylate Complex Pyrophosphate

  7. ATP (adenosine triphosphate) universal energy carrier in all living organisms There is a good correlation between cellular ATP measurement and the number of viable bacteria present.

  8. Previous Study by Univ. of Michigan Sampling locations of drinking water

  9. Prediction of Bacterial Level Water Utilities & Laboratories After 7 days HPC Water Samples Expected HPC HPC UM : ATP assay

  10. A portable microluminometer used in previous study by U of M

  11. ATP assay: Drinking Water ATP vs. plate count United States

  12. ATP assay: Drinking Water ATP vs. plate count Worldwide water samples

  13. Summary of Previous Study • Bacterial Estimation Within Minutes • Real-Time Information • Control Action • High Correlations with HPC • Simplicity • On-Site Application

  14. Reagents • ATP assay mix (luciferin-luciferase)  5ml • ATP assay mix dilution buffer (MgSO4, DTT, EDTA, BSA & tricine buffer)  50ml • ATP standard solution (1 mg/ml, when mixed with 1ml DI)  1ml • Equipment: Luminos (Lab_Bell Inc.,Shawinigan, Quebec, Canada)

  15. Manufacturer’s Procedure 1. Filter 250 – 1000 ml of water 2. Transfer the filter membrane in a sterile test tube 3. Add 5 ml of ATP assay mix dilution buffer (or DI water) 4. Boil for 90 sec 5. Withdraw 0.3 ml of the sample and put in a cuvette 6. Measure luminescence after adding 0.1ml of the ATP assay mix (LL).

  16. Preliminary tests • Determine Gain • Determine wavelength • Determine filtration volume • Is boiling the best way of ATP extraction? • Glass tube vs. disposable tube • Use E. coli spiked water samples • Background RLU • Correlation between RLU and CFU

  17. Glass tube vs. disposable tube

  18. ATP extraction method: Boiling vs. Bacterial releasing agent

  19. More tests • Use of SRA to remove non-bacterial ATP and conditioning luminescence reaction • Optimal Gain: 128, • Optimal Wavelength: λ=420

  20. Water from MI Water Plants • 80% cases showed that 500ml filtration produced lower RLU than 100ml filtration * Higher RLU values among duplicates, ** when filtration vol. is 100ml

  21. Conclusions • Determination of bacterial ATP in drinking water using filtration-based luminescence • Manufacturer’s procedure did not work • Modified procedure - optimal gain, wavelength - Disposable tube - ATP extraction: BRA - Filtration volume: 100ml or less

  22. Acknowledgements Thank the Michigan Section AWWA Special Project Committee who awarded The Michigan Section Laboratory Practice Committee (LPC) Rapid ATP Bacterial Study Also want to thank the 12 Michigan Utilities who participated in this study

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