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  • ATP Bioluminescence for Monitoring

    Drinking Water Quality

    School of Public HealthUniversity of Michigan

    August 2005

    JiYoung Lee & Carmen DumasRolf A. Deininger & Janice Skadsen

    http://images.google.com/imgres?imgurl=www.environmental-center.com/consulting/iatl/faucet1.jpg&imgrefurl=http://www.environmental-center.com/consulting/iatl/iatl.htm&h=204&w=174&prev=/images%3Fq%3Ddrinking%2Bwater%26svnum%3D10%26hl%3Den%26lr%3D%26ie%3DUTF-8%26oe%3DUTF-8%26sa%3DG

  • 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.

  • Michigan Section Laboratory Practice Committee

    Special Projects GrantATP Luminos

  • 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)

  • Ideal Analysis

    Rapid Sensitive Quantitative Differentiate between dead and viable Comparable with current methods Field applicable Simple Cost effective

  • Luciferin

    + ATP

    + PP

    O2

    ATP bioluminescence

    Luciferyl Adenylate Complex

    Pyrophosphate

    Luciferase Mg2+

    Oxyluciferin

    h

    http://www.biotium.com/structures/10100small.gif

  • 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.

  • Hungary

    Switzerland Germany

    U.K. AustriaNetherlands

    France

    China

    Korea

    JapanEgypt

    Australia

    UkraineLithuania

    Saudi Arabia

    Canada

    Argentina

    Peru

    Venezuela

    Brazil

    Panama

    Italy

    New Zealand

    Norway

    South Africa

    Spain

    Thailand

    Vietnam

    Sampling locations of drinking water

    Previous Study by Univ. of Michigan

  • Water Utilities & Laboratories

    UM : ATP assay

    Water Samples Expected HPC

    After 7 days

    HPC

    HPC

    Prediction of Bacterial Level

  • A portable microluminometer used in previous study by U of M

  • l og HP C = 1 . 7 4 * l og ATPr = 0 . 9 3

    0

    1

    2

    3

    4

    5

    6

    7

    8

    0 1 2 3 4 5

    log ATP (RLU/ml)

    log

    HPC

    (CFU

    /ml)

    ATP assay: Drinking WaterATP vs. plate count

    United States

  • log HPC = 1.68 * log ATP r = 0.86

    0

    1

    2

    3

    4

    5

    6

    7

    0 1 2 3 4 5

    log ATP (RLU/ml)

    log

    HPC

    (CFU

    /ml)

    ATP assay: Drinking WaterATP vs. plate count

    Worldwide water samples

  • Summary of Previous Study

    Bacterial Estimation Within Minutes Real-Time Information Control Action

    High Correlations with HPC Simplicity On-Site Application

  • 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)

  • 1. Filter 250 1000 ml of water2. Transfer the filter membrane in a sterile test

    tube3. Add 5 ml of ATP assay mix dilution buffer

    (or DI water)4. Boil for 90 sec5. Withdraw 0.3 ml of the sample and put in a

    cuvette6. Measure luminescence after adding 0.1ml of the

    ATP assay mix (LL).

    Manufacturers Procedure

  • 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

  • Glass tube vs. disposable tube

    Summary Glass tube Plastic tube

    Mean 218632 146006 S.D. 42294 37372

    Range 491392 87274 Minimum 23898 101441 Maximum 515290 188715

  • Summary boiling BRA

    Mean 146006 25047 S.D. 77535 15201

    Range 255492 42582 Minimum 32222 5370 Maximum 287714 47952

    ATP extraction method:Boiling vs. Bacterial releasing agent

  • More tests

    Use of SRA to remove non-bacterial ATP and conditioning luminescence reaction

    Optimal Gain: 128, Optimal Wavelength: =420

  • Water from MI Water Plants

    80% cases showed that 500ml filtration produced lower RLU than 100ml filtrationcity source type RLU * RLU ** pH TH CFU/ml

    St. Clair GW 132176 132176 7.1 114 444Battle Creek GW 92966 92966 7.6 314 291

    Lansing GW 1629 0 9.4 96 62Mt.Pleasant GW 3638 3638 9.3 123 37

    Lapeer SW 13321 13321 124Grand Haven SW 21177 21177 7.6 144 40

    Escanaba SW 9884 9884 9Grand Rapids SW 21832 21832 7.5 142 28

    Bay City SW 13693 13693 8.6 102 37Marquette SW 1399 0 8.3 48 56

    * Higher RLU values among duplicates, ** when filtration vol. is 100ml

  • Conclusions

    Determination of bacterial ATP in drinking water using filtration-based luminescence

    Manufacturers procedure did not work Modified procedure

    - optimal gain, wavelength- Disposable tube- ATP extraction: BRA- Filtration volume: 100ml or less

  • Acknowledgements

    Thank the Michigan Section AWWASpecial Project Committee who awardedThe Michigan Section Laboratory Practice

    Committee (LPC) Rapid ATP Bacterial Study

    Also want to thank the 12 Michigan Utilities who participated in this study

    ATP Bioluminescence for Monitoring Drinking Water QualityScope of ProjectMichigan Section Laboratory Practice CommitteeProblems of Current Test MethodsIdeal AnalysisSummary of Previous StudyReagentsPreliminary testsMore testsWater from MI Water PlantsConclusionsAcknowledgements

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