# Presentation: 546164

## ATP Bioluminescence for Monitoring Drinking Water Quality

**School of Public Health**
**University of Michigan**
**August 2005**

***JiYoung Lee & Carmen Dumas***
***Rolf A. Deininger & Janice Skadsen***

## 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 Grant
- ATP Luminos

- Michigan Section
- American Water Works Association

## 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

## ATP bioluminescence

**Luciferin**

**+ ATP**

- +  PP

- O2

**Luciferyl Adenylate **
**Complex  **

**Pyrophosphate**

**Luciferase   Mg****2+**

**Oxyluciferin**

**h******

**Notes:**
When ATP from bacteria is mixed with Luciferin-Luciferace (enzyme and substate) it goes through a series of reactions and produces light. The amount of light produced is proportional to the amount of ATP. Luciferin-Luciferace is extracted from fireflies.

## 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.

## Sampling locations of drinking water

- Previous Study by Univ. of Michigan

## Prediction of Bacterial Level 

***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

## ATP assay: Drinking Water

- ATP vs. plate count

- United States

## ATP assay: Drinking Water

- ATP 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

## Slide 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)

- 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).

**Manufacturer’s Procedure **

## Slide 17

**Notes:**
Filtration of water samples using a sterile disposable filtration device

## Slide 18

**Notes:**
Treatment with Somatic cell releasing agent to remove non-bacterial ATP. The SRA also do conditioning.

## Slide 19

**Notes:**
Transfer the filtered membrane into a sterile disposable test tube before ATP extraction

## 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

**Notes:**
Plastic tube worked better. Less SD

## ATP extraction method:

- Boiling vs. Bacterial releasing agent

**Notes:**
BRA works better.

## More tests

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

## 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

**Notes:**
Filtration of 500ml of water, which is the manufacturer’s procedure, gave lower RLU values than the filtration volume of 100ml.  It may be due to the accumulation of interference when high volume of water is filtered. 

## Slide 25

## 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

## 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