Introduction to the application of redox-potential measurement in microbiological testing, including MPN. Calibration and validation characteristics are shown. Advantages of this method: * Very simple measurement technique. * It does not require strict temperature control. * Rapid method, especially in the case of high contamination. * Applicable for every nutrient broth (impedimetric methods require special substrates with low conductance). * Especially suitable for the evaluation of the membrane filter methods. * Economic, effective and simple method for heat destruction measurements. * Effective tool for the optimization of the nutrient media. * The test costs are less than those of the classical methods, especially in the case of zero tolerance in quality control (coliforms, Enterococcus, Pseudomonas, etc.).

1. REDOX-POTENTIAL MEASUREMENT
AS A RAPID METHOD
FOR MICROBIOLOGICAL TESTING

2. Problems in microbiological
quality control
Classical methods
Long incubation time (1-4 days)
The applicability, reliability and test price of the
methods are concentration-depending:
High concentration: dilution and colony
counting in the range of
30-300 cfu/ml.
Low concentration: MPN method
Membrane filtering

3. Redox-potential measurement
Physico-chemical base
Assuming a chemical reaction:
a A + b B c C + d D
[C]c [D]d
Q = ------------
[A]a [B]b

4. Free energy and electric work
DG = DG° + R T ln Q
DG = - n FDE.
n F DE = - n F DE° + R T ln Q

5. Electromotive force
R T [C]c [D]d
DE = DE° - ------- ln ---------
n F [A]a [B]b

6. In biological systems
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]

7. The electric effect of the changing could be expressed
by the Nernst equation:
RT [oxidant] [H+]
Eh = E0 + ------ ln ----------------
nF [reductant]
RT [reductant]
Eh = E0 - ------ ln ----------------
nF [oxidant] [H+]
Where Eh is the redox-potential referring to the normal
hydrogen electrode (V)
E0 is the normal redox-potential of the system (V)
R is the Gas-constant R = 8.314 J/mol K
F is the Faraday constant F = 9.648˙104 C/mol (J/V mol)
n is the number of electrons in the redox system (n=1)

8. Test cell for redox potential
measurement

9. Typical redox-curve of the
microbial growth
E. coli 37 °C, TSB
-400
-300
-200
-100
0
100
200
300
400
500
0 1 2 3 4 5 6 7 8 9
t (h)
Eh(mV)
3
4
5
6
7
8
9
lgN
Eh lg N
|dE/dt|>DC
lg Nc
lg N0
TTD

10. The detection time (TTD) is that moment when
the absolute value of the rate of redox potential
change in the measuring-cell overcomes a value
which is significantly differing from the random
changes (e.g. |dE/dt|  0.5 mV/min).
This value is the detection criterion. As the
critical rate of the redox potential decrease
needs a determined cell count the detection time
depends on the initial microbial count.

11. Redox-curves of several bacteria
-400
-300
-200
-100
0
100
200
300
400
500
0 5 10 15 20
t (h)
Eh(mV)
Campylobacter B. subtilis L. monocytogenes
Ent. faecalis Ps. aeruginosa E. coli

12. Effect of the initial Cell-
concentration on the redox-curves
E. coli in TSB
-400
-300
-200
-100
0
100
200
300
400
0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960
t (min)
Eh(mV)
Steril steril lgN=0,09 lgN=2,38
lgN=3,39 lgN=4,25 lgN=4,80
TTD for the redox-potential measurement is: |DE/D t|>1mV/min

13. Effect of the initial cell
concentration on TTD
E. coli in TSB
0
1
2
3
4
5
6
2 3 4 5 6
lgNo (cfu/inoculum)
TTD(h)

14. Determination of calibration
curves
1. External calibration curve
Known microflora
The equation of the calibration curve is
calculated by linear regression from the log
N (determined by classical cultivation) and
the TTD (is determined instrumentally)

15. Determination of calibration
curves
2. Internal calibration curve
Unknown microflora
This method is applied when the composition of the
microflora is not known and previously constructed
calibration curve cannot be taken. In this case, the
redox potential measurement is combined with the
MPN method. Based on the last dilution levels still
showing multiplication, the initial viable count is
calculated using the MPN-table. Based on the
obtained microbe count and TTD values, the internal
calibration curve can be constructed.

16. Determination of the internal
calibration curve 1.

17. Determination of the internal
calibration curve 2.

18. Determination of the internal
calibration curve 3.

19. Validation of the Redox-potential
measuring method

20. Test microorganisms and culture
media of the tests 1.
Microorganisms Redox
potential
Plate
counting
Escherichia coli BBL, TSB TSA, Tergitol
Enterobacter
aerogenes
BBL, TSB TSA, Tergitol
Citrobacter freundii BBL, TSB TSA, Tergitol
Klebsiella oxytoca BBL, TSB TSA, Tergitol
Acinetobacter lwoffii BBL, TSB TSA, Tergitol
Pantoea
agglomerans
BBL, TSB TSA, Tergitol

21. Test microorganisms and culture
media of the tests 2.
Microorganisms Redox
potential
Plate
counting
Pseudomonas
aeruginosa
Cetrimide,
TSB
TSA,
Cetrimide
Pseudomonas
fluorescens
Cetrimide,
TSB
TSA,
Cetrimide
Enterococcus
faecalis
Azide, TSB TSA, Slanetz-
Bartley
Total count TSB TSA

22. Validation characteristics of the
method 1.
Selectivity
it depended on the media used for
identification.
Linearity
from 1 to 107cfu/test flask.

23. Validation characteristics of the
method 2.
Sensitivity
Detection limit
1 cell/test flask.
Quantitation limit
The theoretical quantitation limit is 10 cell/inoculum
(1 log unit), which is in agreement with the
obtained calibration curves.
min13060
Nlg
TTD




24. Validation characteristics of the
method 3.
Range
On the base of the calibration curves the range
lasted from 1 to 7 log unit. Below 10 cells the
Poisson-distribution causes problems, over 107
cells the TTD is too short comparing to the
transient processes (temperature-, redox-
equilibrum, lag-period of the growth).
Repeatability
Calculated from the calibration curves:
SDlgN = 0.092
SDN = 100.092 = 1.24 = 24%

25. Validation characteristics of the
method 4.
Robustness
The most important parameter is the
temperature, which has a double effect on the
results – the growth rate of the microorganisms
and the measured redox-potential are
temperature depending. Performing the
measurements at the temperature optimum of
microorganisms, the growth rate in a ±0.5 °C
interval does not change. The effect of the
temperature on the measured redox-potential
was determined experimentally. The results
showed that the effect of the temperature
variation is negligible.

26. Advantages of the redox-
potential measurement 1.
Very simple measurement technique.
It does not require strict temperature control.
Rapid method, especially in the case of high
contamination.
Applicable for every nutrient broth (impedimetric
methods require special substrates with low
conductance).
Especially suitable for the evaluation of the
membrane filter methods.

27. Advantages of the redox-
potential measurement 2.
Economic, effective and simple method for
heat destruction measurements.
Effective tool for the optimization of the
nutrient media.
The test costs are less than those of the
classical methods, especially in the case
of zero tolerance in quality control
(coliforms, Enterococcus, Pseudomonas,
etc.).

28. Application of the redox method
1. Quality control
Foods
Water
Surfaces
2. Heat destruction of bacteria
3. Activity of bacteria
4. Media optimization
5. Efficiency of disinfectants

29. Quality control 1.
Foods
Enterobacter and total count in raw milk
Nyerstej, 1/2 TSB (T=30 °C)
-400
-300
-200
-100
0
100
200
300
400
500
0 5 10 15 20 25
t (h)
Eh(mV)
0. hig. 1. hig. 2. hig. 3. hig. 4. hig
5. hig 6. hig 7. hig.

30. Quality control 1.
Foods
Enterobacter and total count in raw milk
Nyerstej belső kalibrációs görbe
(1/2 TSB, T=30 °C)
y = 2,6486x + 1,34
R
2
= 0,9895
0
5
10
15
20
0 1 2 3 4 5 6 7
hígítás
TTD(h)
Összcsira Enterobacter
MPNEnterob.=2,3x102
/ml
MPNÖsszcsíra=2,3x106
/ml

31. Comparison of external and
internal calibration curves
Raw milk
y = -1.5014x + 15.413
R2
= 0.9596
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8 9
lgN /ml milk
TTD(h)
Internal External

32. Method time comparison
Sample
Classical method Redox method
lgN Needed
time (h)
lg MPN Needed
time(h)
1. 5,18 5,36
2. 5,06 5,36
3. 4,93 72 4,36 18
4. 6,35 6,36
5. 6,79 6,36

33. Quality control 2.
Water
E. coli in still water
Escherichia coli
0
1
2
3
lgN(cfu/100ml)
MicroTester Plate
1. 1. 2. 2. 3. 3. 4. 4.

34. Quality control 2.
Water
Enterococcus in still water
Enterococcus
0
1
2
3
lgN(cfu/100ml)
MicroTester Plate
1. 1. 2. 2. 3. 3.

35. Method time comparison
Cell count Time needed (h)
(cfu/ 100 ml) Mikroplate Redox
(with membrane
filtering of 100 ml )
Escherichia coli 256
389
310
618
36
7,67
7,17
7,50
6,50
Enterococcus 44
203
219
36
11,79
11,00
10,96

36. Quality control 3.
Surfaces
Redox curves, table surface, TSB, 30°C
-400
-200
0
200
400
600
0 5 10 15 20 25
t (h)
Eh(mV)
0. 1.
2.
3.
Enterobacterium: MPN=2.3∙101
Total count: MPN=2.3∙102

37. Quality control 3.
– The microflora present on the swab is directly
measurable without washing. There is no statistically
significant difference between the microbial counts
obtained with redox-potential measurements and the
plating method.
– By help of internal calibration curve, the viable count
of surfaces with unknown microflora may also be
determined. In further studies of surfaces with
identical microflora, the already established
calibration curve may be applied as an external
calibration curve. Observing the shape of the redox-
curves both the total count and Enterobacterial count
can be determined simultaneously, applying non
selective nutrient broth (TSB) in a single, common
measurement system.

38. Quality control 3.
– Comparing the time requirement of the methods, the
traditional plating method demands 3 days for the
determination of total count while by the redox
method, using internal calibration and depending on
the level of surface contamination, the viable count
can be determined within 15-20 hours or using
external calibration curve (depending on the level of
the surface contamination) it may be determined
within 4-8 hours.
– Applying external calibration curve, when washing of
swabs and the preparation of dilution series are not
necessary, the duration of the examination, the
material, tool and labor requirements can significantly
be reduced.

39. Applications 2.
Heat destruction of bacteria
– Campylobacter jejuni

40. Typical changes in redox-
potential

41. Calibration diagrams
Campylobacter in different selective broths y = -176,56x + 2026,1
R
2
= 0,9738
0
200
400
600
800
1000
1200
1400
1600
1800
2 3 4 5 6 7 8

42. Heat destruction experiments
3 different models:
Classical isotherm model
Redox isotherm model
Redox anisotherm model

43. Thermal death curve –
Classical isotherm method
Classical isotherm
thermal death curve y = -0,086x + 5,3621
R2
= 0,9987
-0,5
0
0,5
1
1,5
48 53 58 63
T (°C)
lgD
Z=11.62°C

44. Thermal death curve –
Redox isotherm method
Thermal death curve y = -0,1012x + 6,2336
R2
= 0,954
-0,5
0
0,5
1
1,5
50 52 54 56 58 60 62 64 66
T (°C)
lgD
Z=9.88°C

45. Thermal death curve –
combined isotherm results
Combined thermal death curve y = -0,092x + 5,7014
R2
= 0,971
-0,5
0
0,5
1
1,5
48 53 58 63
T (°C)
lgD
Z=10.86°C

46. Simplified determination of z-
value
Calibration curve: lgN=a-b·TTD
Decimal reduction time:
D=-Δt/ΔlgN= Δt/(b· ΔTTD)
lgD=lgΔt-lgb-lg(ΔTTD)T
From the thermal death curve:
z
1
T
Dlg




47. Simplified determination of z-
value
z
1
T
TTDlg
T
blg
T
tlg
T
Dlg


D





D



lgΔTTD is a linear function of temperature,
from the slope the z-value can be calculated
T
z
1
ATTDlg D

48. Determination of z-value from
anisotherm heat treatment
On the base of calibration curve: z=9.37 °C
Thermal death curve
y = -0,1067x + 5,5218
R2
= 0,9779
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
-0,1
0
54 55 56 57 58 59
T (°C)
lgD

49. Determination of z-value from
anisotherm heat treatment
On the base of TTDs: z=9.37 °C
Anisotherm heat treatment y = 0,1067x - 3,5787
R2
= 0,9779
1,5
1,8
2,1
2,4
2,7
3
54 55 56 57 58 59
Ti(°C)
lgΔTTD

50. Determination of z-value
Classical
isotherm
method
Redox
isotherm
method
Redox
anisotherm
method
z-value (°C)
from 4 points
11.63
R2=0.999
9.88
R2=0.954
9.37
R2=0.978
Substrates
needed
12×6=72
Petri-dishes
(dilution series)
12 test flasks 5 test flasks
Additional
equipment
6 jars and
6 microaerophil
sacks
- -
Incubation
time
48 (96)h 35h 35h

51. Applications 3.
Examination of microbial activity in
soil
–Effects of antibiotics

52. Applications 3.
Effect of doxycyline (T1 – T5: soil types)
Doxycycline
y = 8.922x
R
2
= 0.9943
y = 6.8416x
R
2
= 0.9498
y = 4.5039x
R
2
= 0.9772
y = 13.544x
R
2
= 0.9835
y = 2.1526x
R2
= 0.9568
0
2
4
6
8
10
12
14
16
18
0 1 2 3
lgc-lgco
TDT-TDTo
T1 T2 T3 T4 T5