MicroTester as a validated method is suitable for rapid microbiological testing of mineral water, carbonated water, tank and running drinking water and other types of water. The time needed for a reliable detection of microorganisms is of key importance: in water industry the real-time (or at least as fast as possible) monitoring of the microbiological properties of the production is indispensable; in public water supply the essential basis of the epidemiological and public health measures is the fast and reliable result of the microbiological inspection. Beside the most important and most widely inspected microbiological contaminants the most relevant disturbing flora was involved to the validation process as well.

1. Microbiological inspection of
mineral water by redox-potential
measurement
Dr. Olivér Reichart
Dr. Katalin Szakmár

2. Introduction
MicroTester as a validated method is suitable for rapid
microbiological testing of mineral water, carbonated water, tank
and running drinking water and other types of water. The time
needed for a reliable detection of microorganisms is of key
importance: in water industry the real-time (or at least as fast as
possible) monitoring of the microbiological properties of the
production is indispensable; in public water supply the essential
basis of the epidemiological and public health measures is the
fast and reliable result of the microbiological inspection. Beside
the most important and most widely inspected microbiological
contaminants the most relevant disturbing flora was involved to
the validation process as well.

3. Theoretical base
The energy source of the growth is the biological
oxidation which results in a reduction in the
environment.
This is due to the oxygen depletion and the production of
reducing compounds in the nutrient medium.
A typical oxidation-reduction reaction in biological
systems:
[Oxidant] + [H+] + n e- [Reductant]

4. A typical redox curve of the
microbial growth
DC: Detection Criterion
TTD: Time to Detection

5. Microorganisms
The most frequently tested contaminant
microorganisms in mineral water productions
are:
Coliforms
Escherichia coli
Pseudomonas aeruginosa
Enterococcus faecalis
Total count (22 °C and 37 °C)

6. Method validation
Selectivity
Linearity
Sensitivity
Detection limit
Repeatability
Robustness

7. Selectivity 1.
Coliforms and Acinetobacter lwoffii in BBL.
(K.o.: Klebsiella oxytoca, Ent.: Enterobacter aerogenes, Citro.: Citrobacter freundii,
E.c.: Escherichia coli, Acin.: Acinetobacter lwoffii)
Coliforms and Acinetobacter in BBL
-400
-200
0
200
400
600
0 5 10 15 20
t (h)
Eh(mV)
K.o.lgN=3,55 Citro lgN=3 Ent lgN=3,48
E.c. lgN=3,67 Acin. lgN=3,65

8. Selectivity 2.
Micrococcus and Enterococcus in Azid broth
Enterococcus and Micrococcus
0
50
100
150
200
250
300
350
400
0 5 10 15 20
t (h)
Eh(mV)
Enterococcus Micrococcus

9. Selectivity 3.
Growth in Cetrimide broth
0
100
200
300
400
500
0 5 10 15 20 25
t (min)
Eh(mV)
Ps. aeruginosa Ps. fluoresc. E. coli Enterococcus
Pseudomonas aeruginosa, Pseudomonas fluorescens,
E. coli and Enterococcus faecalis in Cetrimid broth.

10. Linearity
The linear relationship between the logarithm of the
cell concentration and TTD values is demonstrated by
the calibration curves. From the concentrated
suspensions of the test microorganisms tenfold dilution
series were prepared in physiological salt solution. From
the members of the dilution series the redox-potential
test flasks were inoculated with 1.0 ml suspension and
the TTD values were determined.

11. Linearity
Calibration curves of Coliforms
Coliforms in BBL
y = -1.699x + 17.004
R2
= 0.9958
y = -1.471x + 14.26
R2
= 0.9714
y = -1.3506x + 12.896
R2
= 0.9941
y = -1.1775x + 10.184
R2
= 0.9907
0
5
10
15
20
0 1 2 3 4 5 6 7
lgN (cfu/cell)
TTD(h)
Citrobacter Klebsiella oxytoca Enterobacter E. coli (37 °C)

12. Linearity
Calibration curve of E. coli
Escherichia coli
y = -0.8393x + 7.1607
R2
= 0.9988
0
2
4
6
8
0 1 2 3 4 5 6 7
logN/100 ml
TTD(h)

13. Linearity
Calibration curve of Enterococcus faecalis
Enterococcus Azid
y = -1.5873x + 13.222
R
2
= 0.9859
0
5
10
15
0 1 2 3 4 5 6 7 8
logN/100ml
TTD(h)

14. Linearity
Calibration curve of Pseudomonas aeruginosa
Pseudomonas aeruginosa Cetrimid
y = -2.5536x + 23.709
R2
= 0.9882
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8
logN/100ml
TTD(h)

15. Linearity
Total count (37 °C)
y = -1.0135x + 8.7505
R2
= 0.9973
0
2
4
6
8
10
0 1 2 3 4 5 6 7 8
logN/100ml
TTD(h)
Calibration curve of total count

16. Sensitivity
Microorganism Broth Regression equation Sensitivity
(min/log
unit)
Citrobacter freundii BBL TTD (min) = 1190 - 132·lgN 132
Klebsiella oxytoca BBL TTD (min) = 856 – 88·lgN 88
Enterobacter aerogenes BBL TTD (min) = 774 – 81·lgN 81
Escherichia coli BBL TTD (min) = 596 – 68·lgN 68
Pseudomonas aeruginosa Cetrimid TTD (min) = 1440 – 155·lgN 155
Enterococcus faecalis Azid TTD (min) = 836 – 92·lgN 92
The sensitivity of the measuring method was
determined as the slope of the calibration curves.

17. Detection limit
 The detection limit is 1 cell/test flask, so the
system is suitable for the absence/presence tests,
so considerable costs and time could be saved
with more membrane filters joined together.
 On the base of the calibration curves the range
lasted from 1 to 7 log unit.

18. Repeatability
The repeatability calculated from the calibration
curves:
SDlgN = 0.092
SDN = 100.092 = 1.24 = 24%
which complies with the requirements of
microbiological methods.

19. Quality control tests
72 bottles tested for Coliform
Testing method of Laboratory
 Membrane filtering of 3x250 ml mineral water with 1
filter. Cultivation Tergitol agar at 37 °C for 48 h. One
Petri dish represents 3 bottles of mineral water.
Redox-potential measurement method
 Membrane filtering of 3x250 ml mineral water with 1
filter, placing 4 membranes into 1 test flask containing
BBL broth. Temperature: 37 °C. One test flask
represents 12 bottles of mineral water.
 Positive control: 1 ml of Citrobacter freundii suspension
(lgN = 3.66)

20. Quality control test
Results of redox-potential measurement of 72 bottles
72 bottles
-400
-300
-200
-100
0
100
200
300
400
500
600
0 2 4 6 8 10 12 14 16 18 20 22 24 26
t (h)
Eh(mV)
1. - 12. 13. - 24. 25. - 36. 37. - 48.
49. - 60. 61. - 72. Citrobacter

21. Quality control test
Bottles 1.-12. 13.-24. 25.-36. 37.-48. 49.-60. 61.-72.
Laboratory negative negative negative negative negative negative
Redox negative negative negative negative negative negative
Results of 72 bottles test

22. 66 bottles tested for Coliforms
Testing method of Laboratory
 Membrane filtering of 3x250 ml mineral water with 1 filter.
Cultivation Tergitol agar at 37 °C for 48 h. One Petri dish
represents 3 bottles of mineral water.
Redox-potential measurement method
 Membrane filtering of 3x250 ml mineral water with 1 filter,
placing 3 membranes into 1 test flask containing BBL broth.
Temperature: 37 °C. One test flask represents 9 bottles of
mineral water.
 Besides the mineral water two technological water samples were
tested for Coliforms
 Positive control: 1 ml of Escherichia coli suspension (lgN = 6.7)

23. Quality control test
Results of redox-potential measurement of 66 bottles
66 bottles
-400
-300
-200
-100
0
100
200
300
400
500
0 2 4 6 8 10 12 14 16 18 20 22 24
t (h)
Eh(mV)
1.-9. 10.-18. 19.-27. 28.-36. 37.-45. 46.-54.
55.-63. 64.-66. E.coli (+) Negativ

24. Quality control test
Samples 1.-66. Bottles Water sample 1. Water sample 2.
Laboratory results negative negative negative
Redox method negative negative negative
Results of 66 bottles test

25. Detection time of one cell
Microbe One cell detection time (h)
Escherichia coli 11
Citrobacter freundii 23
Pseudomonas aeruginosa 24
Enterococcus faecalis 15

26. Results of industrial tests
Microbe
All
measurements
(piece)
Match the
standard
test (%)
False
positive
results
(%)
False
negative
results
(%)
Escherichia
coli
942 99,89 0,11 0,00
Coliform 4674 99,87 0,00 0,13
Enterococcus 3000 99,93 0,00 0,07
Pseudomonas
aeruginosa
3372 99,82 0,06 0,12

27. Advantages of the redox-potential
measurement
 Very simple measurement technique.
 Rapid method, especially in the case of high
contamination.
 Applicable for every nutrient broth
 Especially suitable for the evaluation of the membrane
filter methods.
 The test costs are less than those of the classical
methods, especially in the case of zero tolerance
(Coliforms, Enterococcus, Pseudomonas, etc.).