redox-potential measurement as a rapid method for microbiological testing
TRANSCRIPT
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:
a A + b B c C + d D
[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
R T [C]c [D]d DE = DE° - ------- ln ---------
n F [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
-400
-300
-200
-100
0
100
200
300
400
500
0 1 2 3 4 5 6 7 8 9
t (h)
Eh
(m
V)
3
4
5
6
7
8
9
lg N
Eh lg N
|dE/dt|>DC
lg Nc
lg N0TTD
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
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-100
0
100
200
300
400
500
0 5 10 15 20
t (h)
Eh
(m
V)
Campylobacter B. subtilis L. monocytogenes
Ent. faecalis Ps. aeruginosa E. coli
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: |E/ t|>1mV/min
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)
TT
D (
h)
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 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
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
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
Detection limit
1 cell/test flask.
Quantitation limitThe theoretical quantitation limit is 10
cell/inoculum (1 log unit), which is in agreement with the obtained calibration curves.
min13060Nlg
TTD
Validation characteristics of the method 3.
RangeOn 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).
RepeatabilityCalculated 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 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.
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.).
Application of the redox method
1. Quality controlFoods
Water
Surfaces
2. Heat destruction of bacteria
3. Activity of bacteria
4. Media optimization
5. Efficiency of disinfectants
Quality control 1.Foods
Enterobacter and total count in raw milk
Nyerstej, 1/2 TSB (T=30 °C)
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-100
0
100
200
300
400
500
0 5 10 15 20 25
t (h)
Eh
(m
V)
0. hig. 1. hig. 2. hig. 3. hig. 4. hig
5. hig 6. hig 7. 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
R2 = 0,9895
0
5
10
15
20
0 1 2 3 4 5 6 7
hígítás
TT
D (
h)
Összcsira Enterobacter
MPNEnterob.=2,3x102/ml
MPNÖsszcsíra=2,3x106/ml
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
TT
D (
h)
Internal External
Method time comparison
SampleClassical 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
Quality control 2.Water
E. coli in still water
Escherichia coli
0
1
2
3
lgN
(cf
u/1
00 m
l)
MicroTester Plate
1. 1. 2. 2. 3. 3. 4. 4.
Quality control 2.Water
Enterococcus in still water
Enterococcus
0
1
2
3
lgN
(cf
u/1
00 m
l)
MicroTester Plate
1. 1. 2. 2. 3. 3.
Method time comparisonMethod time comparison
Cell count Time needed (h)
(cfu/ 100 ml) Mikroplate Redox(with membrane
filtering of 100 ml )
Escherichia coli 256 389 310 618
367,677,177,506,50
Enterococcus 44 203 219
3611,7911,0010,96
Quality control 3.
SurfacesRedox curves, table surface, TSB, 30°C
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0
200
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600
0 5 10 15 20 25
t (h)
Eh
(m
V)
0. 1.
2.
3.
Enterobacterium: MPN=2.3∙101
Total count: MPN=2.3∙102
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.
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 redox-potential
Calibration diagrams
Campylobacter in different selective broths y = -176,56x + 2026,1
R2 = 0,9738
0
200
400
600
800
1000
1200
1400
1600
1800
2 3 4 5 6 7 8
Heat destruction experiments
3 different models:
Classical isotherm modelRedox isotherm modelRedox anisotherm model
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
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
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
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
Simplified determination of z-value
z
1
T
TTDlg
T
blg
T
tlg
T
Dlg
lgΔTTD is a linear function of temperature, from the slope the z-
value can be calculated
Tz
1ATTDlg
Determination of z-value from anisotherm heat treatment
On the base of calibration curve: z=9.37 °C
Thermal death curvey = -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
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)
lgΔ
TT
D
Determination of z-valueClassical isotherm method
Redox isotherm method
Redox anisotherm
method
z-value (°C) from 4 points
11.63R2=0.999
9.88R2=0.954
9.37R2=0.978
Substrates needed
12×6=72 Petri-dishes
(dilution series)
12 test flasks 5 test flasks
Additional equipment
6 jars and6 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)
Doxycycliney = 8.922x
R2 = 0.9943
y = 6.8416x
R2 = 0.9498
y = 4.5039x
R2 = 0.9772
y = 13.544x
R2 = 0.9835
y = 2.1526x
R2 = 0.9568
02468
1012141618
0 1 2 3lgc-lgco
TDT-
TDTo
T1 T2 T3 T4 T5