Redox-potential measurement as a rapid method for microbiological testing

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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0

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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

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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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0

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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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600

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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