REDOX-POTENTIAL MEASUREMENT
AS A RAPID METHOD
FOR MICROBIOLOGICAL TESTING
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
Redox-potential measurement
Physico-chemical base
Assuming a chemical reaction:
aA+bB
cC+dD
[C]c [D]d
Q = -----------[A]a [B]b
Free energy and electric work
DG = DG° + R T ln Q
DG = - n FDE.
n F DE = - n F DE° + R T ln Q
Electromotive force
RT
[C]c [D]d
DE = DE° - ------- ln --------nF
[A]a [B]b
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]
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)
Test cell for redox potential
measurement
Typical redox-curve of the
microbial growth
E. coli 37 °C, TSB
Eh
lg N
500
9
400
8
|dE/dt|>DC
200
7
lg Nc
100
6
0
-100
5
-200
-300
lg N0
-400
0
1
2
3
4
TTD
3
4
5
t (h)
6
7
8
9
lg N
Eh (mV)
300
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.
Redox-curves of several bacteria
500
400
300
Eh (mV)
200
100
0
-100
-200
-300
-400
0
5
10
15
t (h)
Campylobacter
B. subtilis
L. monocytogenes
Ent. faecalis
Ps. aeruginosa
E. coli
20
Effect of the initial Cellconcentration on the redox-curves
E. coli in TSB
400
300
Eh (mV)
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)
Steril
lgN=3,39
steril
lgN=4,25
lgN=0,09
lgN=2,38
lgN=4,80
TTD for the redox-potential measurement is: |DE/D t|>1mV/min
Effect of the initial cell
concentration on TTD
E. coli in TSB
6
TTD (h)
5
4
3
2
1
0
2
3
4
lgNo (cfu/inoculum)
5
6
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)
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.
Determination of the internal
calibration curve 1.
Determination of the internal
calibration curve 2.
Determination of the internal
calibration curve 3.
Validation of the Redox-potential
measuring method
Test microorganisms and culture
media of the tests 1.
Microorganisms
Escherichia coli
Enterobacter
aerogenes
Citrobacter freundii
Klebsiella oxytoca
Acinetobacter lwoffii
Pantoea
agglomerans
Redox
Plate
potential
counting
BBL, TSB TSA, Tergitol
BBL, TSB TSA, Tergitol
BBL, TSB
BBL, TSB
BBL, TSB
BBL, TSB
TSA, Tergitol
TSA, Tergitol
TSA, Tergitol
TSA, Tergitol
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, SlanetzBartley
Total count
TSB
TSA
Validation characteristics of the
method 1.
Selectivity
it depended on the media used for
identification.
Linearity
from 1 to 107cfu/test flask.
Validation characteristics of the
method 2.
Sensitivity
TTD
 60  130 min
 lg N
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.
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-, redoxequilibrum, lag-period of the growth).
Repeatability
Calculated from the calibration curves:
SDlgN = 0.092
SDN = 100.092 = 1.24 = 24%
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.
Advantages of the redoxpotential 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.
Advantages of the redoxpotential 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.).
Application of the redox method
1.
Quality control
Foods
Water
Surfaces
Heat destruction of bacteria
3. Activity of bacteria
4. Media optimization
5. Efficiency of disinfectants
2.
Quality control 1.
Foods
Enterobacter and total count in raw milk
Eh (mV)
Nyerstej, 1/2 TSB
(T=30 °C)
500
400
300
200
100
0
-100
-200
-300
-400
0
5
10
15
20
25
t (h)
0. hig.
1. hig.
2. hig.
5. hig
6. hig
7. hig.
3. hig.
4. hig
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
2
R = 0,9895
20
MPNÖsszcsíra=2,3x106/ml
TTD (h)
15
10
MPNEnterob. =2,3x102/ml
5
0
0
1
Összcsira
2
3
Enterobacter
4
5
6
7
hígítás
Comparison of external and
internal calibration curves
Raw milk
14
y = -1.5014x + 15.413
12
R2 = 0.9596
TTD (h)
10
8
6
4
2
0
1
2
Internal
3
External
4
5
6
7
8
9
lgN /ml milk
Method time comparison
Classical method
Redox method
Sample
lgN
1.
5,18
5,36
2.
5,06
5,36
3.
4,93
4.
6,35
6,36
5.
6,79
6,36
Needed
time (h)
72
lg MPN
4,36
Needed
time(h)
18
Quality control 2.
Water
E. coli in still water
Escherichia coli
lgN (cfu/100 ml)
3
2
1
1.
1.
2.
2.
3.
3.
0
MicroTester
Plate
4.
4.
Quality control 2.
Water
Enterococcus in still water
Enterococcus
lgN (cfu/100 ml)
3
2
1
1.
1.
2.
2.
MicroTester
Plate
0
3.
3.
Method time comparison
Cell count
(cfu/ 100 ml)
Escherichia coli 256
389
310
618
Enterococcus
44
203
219
Time needed (h)
Mikroplate
36
36
Redox
(with membrane
filtering of 100 ml )
7,67
7,17
7,50
6,50
11,79
11,00
10,96
Quality control 3.
Surfaces
Redox curves, table surface, TSB, 30°C
600
3.
Eh (mV)
400
Total count: MPN=2.3∙102
200
2.
0
0.
-200
1.
Enterobacterium: MPN=2.3∙101
-400
0
5
10
15
t (h)
20
25
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 redoxcurves both the total count and Enterobacterial count
can be determined simultaneously, applying non
selective nutrient broth (TSB) in a single, common
measurement system.
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.
Applications 2.
Heat destruction of bacteria
– Campylobacter jejuni
Typical changes in redoxpotential
Calibration diagrams
Campylobacter in different selective broths
y = -176,56x + 2026,1
2
R = 0,9738
1800
1600
1400
1200
1000
800
600
400
200
0
2
3
4
5
6
7
8
Heat destruction experiments
3 different models:
 Classical isotherm model
 Redox isotherm model
 Redox anisotherm model
Thermal death curve –
Classical isotherm method
Classical isotherm
thermal death curve
y = -0,086x + 5,3621
R2 = 0,9987
1,5
lgD
1
0,5
0
-0,5
48
53
58
T (°C)
Z=11.62°C
63
Thermal death curve –
Redox isotherm method
Thermal death curve y = -0,1012x + 6,2336
R2 = 0,954
1,5
lgD
1
0,5
0
-0,5
50
52
54
56
58
T (°C)
Z=9.88°C
60
62
64
66
Thermal death curve –
combined isotherm results
y = -0,092x + 5,7014
R2 = 0,971
Combined thermal death curve
1,5
lgD
1
0,5
0
-0,5
48
53
58
T (°C)
Z=10.86°C
63
Simplified determination of zvalue
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:
 lg D
1

T
z
Simplified determination of zvalue
 lg D  lg Dt  lg b  lg DTTD
1




T
T
T
T
z
1
lg DTTD  A   T
z
lgΔTTD is a linear function of temperature,
from the slope the z-value can be calculated
Determination of z-value from
anisotherm heat treatment
On the base of calibration curve:
Thermal death curve
z=9.37 °C
y = -0,1067x + 5,5218
R2 = 0,9779
0
-0,1
-0,2
lgD
-0,3
-0,4
-0,5
-0,6
-0,7
-0,8
54
55
56
57
T (°C)
58
59
Determination of z-value from
anisotherm heat treatment
On the base of TTDs:
z=9.37 °C
Anisotherm heat treatment
3
y = 0,1067x - 3,5787
R2 = 0,9779
lgΔTTD
2,7
2,4
2,1
1,8
1,5
54
55
56
57
Ti(°C)
58
59
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
Applications 3.
Examination of microbial activity in
soil
– Effects of antibiotics
Applications 3.
Effect of doxycyline (T1 – T5: soil types)
Doxycycline
TDT-TDTo
y = 8.922x
18
16
14
12
10
8
6
4
2
0
2
R = 0.9943
y = 6.8416x
2
R = 0.9498
y = 4.5039x
2
R = 0.9772
y = 13.544x
2
R = 0.9835
0
1
2
3
lgc-lgco
T1
T2
T3
T4
y = 2.1526x
T5
R2 = 0.9568