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 [C] c [D] d Q = ------------ [A] a [B] b [A] a [B] b

4. Free energy and electric work  G =  G° + R T ln Q  G = - n F  E. n F  E = - n F  E° + R T ln Q

5. Electromotive force R T [C] c [D] d R T [C] c [D] d  E =  E° - ------- ln --------- n F [A] a [B] b 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 + ] RT [oxidant] [H + ] E h = E 0 + ------ ln ---------------- nF [reductant] nF [reductant] RT [reductant] RT [reductant] E h = E 0 - ------ ln ---------------- nF [oxidant] [H + ] nF [oxidant] [H + ] Where E h is the redox-potential referring to the normal hydrogen electrode (V) E 0 is the normal redox-potential of the system (V) E 0 is the normal redox-potential of the system (V) R is the Gas-constantR = 8.314 J/mol K R is the Gas-constantR = 8.314 J/mol K F is the Faraday constantF = 9.648˙10 4 C/mol (J/V mol) F is the Faraday constantF = 9.648˙10 4 C/mol (J/V mol) n is the number of electrons in the redox system (n=1) 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

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

12. Effect of the initial Cell- concentration on the redox-curves TTD for the redox-potential measurement is: |  E/  t|>1mV/min

13. Effect of the initial cell concentration on TTD

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 TSBTSA

22. Validation characteristics of the method 1. Selectivity it depended on the media used for identification. Linearity from 1 to 10 7 cfu/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.

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 10 7 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: SD lgN = 0.092 SD N = 10 0.092 = 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 FoodsWaterSurfaces 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 Enterobacter and total count in raw milk

30. Quality control 1. Foods Enterobacter and total count in raw milk Enterobacter and total count in raw milk

31. Comparison of external and internal calibration curves Raw milk

32. Method time comparison Sample Classical method Redox method lgN Needed time (h) lg MPN Needed time(h) 1.5,185,36 2.5,065,36 3.4,93724,3618 4.6,356,36 5.6,796,36

33. Quality control 2. Water E. coli in still water

34. Quality control 2. Water Enterococcus in still water

35. Method time comparison Cell count Time needed (h) (cfu/ 100 ml) (cfu/ 100 ml)MikroplateRedox (with membrane filtering of 100 ml ) Escherichia coli 256 389 389 310 310 618 618367,677,177,506,50 Enterococcus 44 203 203 219 219 36 3611,7911,0010,96

36. Quality control 3. Surfaces

37. –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

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

43. Thermal death curve – Classical isotherm method Z=11.62°C

44. Thermal death curve – Redox isotherm method Z=9.88°C

45. Thermal death curve – combined isotherm results 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:

47. Simplified determination of z- value lgΔTTD is a linear function of temperature, from the slope the z-value can be calculated

48. Determination of z-value from anisotherm heat treatment On the base of calibration curve:z=9.37 °C

49. Determination of z-value from anisotherm heat treatment On the base of TTDs:z=9.37 °C

50. Determination of z-value Classical isotherm method Redox isotherm method Redox anisotherm method z-value (°C) from 4 points 11.63 R 2 =0.999 9.88 R 2 =0.954 9.37 R 2 =0.978 Substrates needed 12×6=72Petri-dishes (dilution series) 12 test flasks 5 test flasks Additional equipment 6 jars and 6 microaerophil sacks -- Incubation time 48 (96)h 35h35h

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

52. Applications 3. Effect of doxycyline (T1 – T5: soil types)