The combination of filtration and ATP+ADP+AMP assay for the assessment of microbial quality of water
Chiaki Hara, Yuko Ichiyanagi, and Shigeya Suzuki, Kikkoman Biochemifa Company
1. Introduction

3. Result

Water microorganisms are a useful indicator for the quality of drinking water, and for the maintenance of water-related facilities. Since
culturing methods take several days, a rapid assay is required for quick response measures for incidents caused by microorganisms.
ATP swabbing tests have been examined for the assessment of microbial quality of water by the detection of ATP from organic debris
including bacteria. However, there are two issues as below.
1. ATP from both microbial and non-microbial sources can be detected, and they cannot be differentiated.
2. The concentrations of ATP in microorganisms can vary during culture; ATP is predominant at the initial stages, but AMP becomes
predominant at later time points (1). Another previous study also showed that ATP+ADP+AMP (A3) in microorganisms is more stable
than ATP, which is affected by nutritional conditions (2).
Based on these points, a rapid and easy test for the assessment of the microbial quality of water was developed; the A3Figure 1.
test (Fig. 1) (3) is employed after sample pretreatment with syringe filter for the elimination of non-microbial A3 andThe luminometer andconcentration of microorganisms (Fig. 2). Moreover, the correlation between new A3 filtration assay and culturing method
the swab of the A3 test.
was evaluated using practical water samples.

3-1. Correlation between RLU and CFU

3-2. Monitoring RLU and CFU over time

The correlation between luminescenceintensity (RLU) based on microbial A3 andviable microbial count (CFU/mL) for eachsample was evaluated.

The quality of water samples over time was evaluated byluminescence unit (RLU) and microbial count (CFU/mL). Fig. 4
shows the RLU () and CFU/mL() for each sample.

Figure 2. Concept and method of new A3 filtration assay

Total operation time for a sample is approximately 3 min.

2. Method
Table
1.
Samplesforassays
2-1. Sample
SampleStorage term (days)
Eight kinds of water samples were collected. In order to obtain samples containing
Well water
0, 5, 11, 18
different microbial quality, a total of 29 water samples were prepared by storing at 25°C for
Utility waste water
0, 5, 11, 18
0-18 days (Table 1).
Water from a water basin for
2, 3, 4, 7, 11, 14
2-2. Viable microbial counthand washing of a shrine
Dilutions of water samples were plated on R2A agar and incubated at 30°C for 2-5 days.
Pure water tank0
Viable microbial counts were measured as colony forming units (CFU).
Cooling tower water A0, 1, 7
Cooling tower water B
0, 5, 11, 18
2-3. Filtration and A3 test
Tap water A
0, 1, 7, 14
Each sample (10 mL) was filtered through a syringe filter (pore size; 0.8 μm, material;
Tap water B
0, 1, 7
cellulose acetate) to eliminate extracellular A3 and trap microorganisms. Successively,
the surfactant was drawn into the syringe using the piston to extract microorganism-derived A3 by the treatment of the filter with the surfactant.
Since the extracted A3 passes through the filter, the syringe piston was pushed down to collect the extracted A3 in a new container. The extracted
A3 was measured by the A3 test (LuciPacTM A3 Surface/LumitesterTM Smart, Kikkoman Biochemifa Company), and the measurement output wasrelative light units (RLU) (Fig. 2).

0112345678log CFU/mLFigure 3. Correlation between RLU andCFU/mL in logarithmic scale
The measurement values for all samples
were 1.2-4.1 log RLU and 2.2-6.3 log
CFU/mL. A positive correlation was foundbetween RLU and CFU/mL in logarithmicscale (y = 0.622x - 0.147, r2 = 0.831).

3221010 15 2000510 15 20days

Cooling tower water A

Tap water A

20y = 0.6217x - 0.1474
R2 = 0.8310

4days

3254151510105501530

1020

5100000

510days0510× 102 RLU

15× 103 RLU

2× 105 CFU/mL

2× 102 RLU

× 104 CFU/mL

300surfactant

4040surfactant

46× 104 CFU/mL

Microbial A3passes throughthe filter.

Dip the cotton swab in
the sample and put itinto the Lucipac tomeasureluminescence using
Lumitester.

5× 102 RLU

Surfactant
extracts
microbial A3.

Push down the piston
to discharge thesample into container.

6× 104 CFU/mL

Microorganisms
are trapped on
the filter.

Use the piston to draw
the surfactant into thesyringe.

Utility waste water
Pure water tank
Cooling tower water B
Tap water B

5log RLU

SamplePut the sample in thesyringe and push down
the piston to filtrate.

Well water
Shrine's water
Cooling tower water A
Tap water A
approximate curve

Utility waste water

Well water15days

Figure 4. Monitoring RLU and CFU over time
During sample storage, the RLU measurements and log CFU
numbers increased over time in sample of cooling tower water
A, In the other samples, the values increased and thendecreased. The trend of consistency between changes of RLUand CFU was confirmed.

4. Summary◆A test with quick and simple procedure was developed by combining the filtration process with the A3 test.
◆A positive correlation was found between the new A3-filtration assay and the culturing method.
◆Changes in viable counts over time could be monitored with the combination of filtration and A3 test.
◆This method is useful for rapid hygiene control of water and water-related facilities.
(1) Smith, N. W., Sindelar, J. J., and Rankin, S. A., J. Food Prot. 2019; 82: 2088–2093.
(2) Sakakibara, T., Murakami, S., and Imai, K., Anal. Biochem. 2003; 312: 48–56.
(3) Bakke, M., and Suzuki, S., J. Food Prot. 2018; 81: 729–737.