Appl Microbiol Biotechnol (2003) 63:3541 DOI 10.

1007/s00253-003-1338-z

ORIGINAL PAPER

T. Ribeiro G. Romestant J. Depoortere A. Pauss

Development, validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control
Received: 20 March 2003 / Revised: 11 April 2003 / Accepted: 11 April 2003 / Published online: 29 May 2003  Springer-Verlag 2003

Abstract We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique. It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells. These measuring cells are individual impedance-meters that can be activated independently of one another. In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 mol of CO2. With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1108 colonyforming units (CFU) ml1. The main use for such devices could be the detection of contamination in foodstuffs. Several of these foodstuffs, when incubated at 37 C, spontaneously release quite large amounts of CO2. Our impedancemeter, however, was able to detect an E. coli presence in canned French beans at 2.35102 CFU ml1 and a S. cerevisiae contamination of apple pure in glass jars at 6.1103 CFU ml1. The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E. coli cultures). The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs. Some industrial assays are in process to complement these laboratory tests. Compared with other available techniques for CO2 measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best
T. Ribeiro A. Pauss ()) Dpartement de Gnie Chimique, Universit de Technologie de Compigne, BP 20529, 60205 Compigne, France e-mail: Andre.Pauss@utc.fr Tel.: +33-344-234457 Fax: +33-344-235216 G. Romestant AES Laboratoire, route de Dol, BP 54, 35270 Combourg, France J. Depoortere SLEL, 107 rue de Klber, 59493 Villeneuve dAscq, France

compromise between sensitivity, technical constraints, and cost. A commercial version of the impedancemeter would enable routine measurements in the quality control of foodstuffs, pharmaceuticals, cosmetics and in R&D laboratories.

Introduction
Measuring impedances can be used in microbiology to perform the detection, quantification, and even identification of some bacteria. Basic knowledge about this subject is provided by Ur and Brown (1975), FirstenbergEden and Eden (1984), and the reviews of Silley and Forsythe (1996) and Wawerla et al. (1999). Two measuring techniques, direct and indirect impedance measurement, are used in microbiology. For more than 30 years, both these techniques and their applications have been widely described; and commercial apparatus has been on the market for about 20 years. At least two French AFNOR (2000, 2002) standards, V08-105 and V08-106, give directives for measuring impedances for quality control in food microbiology and especially for the indirect detection of Escherichia coli in live shellfish. For indirect impedance measurements, the electrodes are immersed in the growing medium and the impedance variations stem from variations in the conductance of the electrolyte, in turn produced by the metabolic activity of the microorganisms (Silley and Forsythe 1996). The indirect technique is used when the salt concentration of the culture broth is either too high or too low; and in this way the microbial metabolism is monitored via the production of CO2 (Owens et al. 1989) and KOH is added to the impedance cell. The inoculated culture medium is located in a chamber separated from the electrodes and the KOH. The CO2 produced as a result of normal metabolism or microbial activity is absorbed by the KOH, causing a decrease in the impedance values. Thus, the variation in impedance is quantitatively and

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qualitatively correlated with CO2 production, according to the following reaction: 2KOH+2CO2!2KHCO3 Consequently, the molar conductance of the reactant solution (DL0), per mole of CO2, is calculated by: DL0=L0(HCO3)L0(OH) DL0=44.5104198.3104 DL0=153.8104 S m2 mol1 The indirect technique is achieved by manually adapted apparatus designed for indirect measurement. Currently, no specific indirect impedancemeter is commercially available. Many articles and papers describe applications of the direct and indirect techniques; and most of them are reviewed by Silley and Forsythe (1996) and Waverla et al. (1999). Most applications focus on the detection and quantification of microorganisms in milk, dairy products, meat, fish, frozen vegetables, grain products, confectionery, yeast in fruit juice, wine, or E. coli in potable water. More specifically, the indirect impedance technique has enabled several applications, like: (1) the detection and estimation of yeast in fruit juice (Deak and Beuchat 1993; Owens 1989), (2) the detection of food-borne bacteria (Bolton 1990), the detection and the estimation of Pseudomonas (Franken and van der Zouwen 1993), Shewanella putrefasciens (Le Den 1995), Salmonella (Madden et al. 1996), E. coli in potable water (Colquhoun et al. 1996), and Haematococcus pluvialis (Gong and Chen 1997) and Clostridium perfringens in foodstuff (Blivet 1997), (3) the detection of specific bacteriophages of lactic bacteria (Svensson 1994), (4) the detection and monitoring of specific activity and microbial growth in heterogeneous samples (Fortin et al. 1996), and (5) the determination of the sensitivity of a specific antibiotic. During this last type of test, it is possible to determine the minimal inhibitory concentration of antibacterial agents (Sawai et al. 2002). In this paper, we describe a new indirect impedance device and some potential applications that were examined during a R&D project in a partnership between the AES Laboratoire, the SLEL Societies, and our laboratory. This impedancemeter is used for measuring growth and/or microbial activity by quantifying the CO2 produced by microorganisms metabolism, without considering the type of media.

incubated in brain heart broth (Institut Pasteur) at 37 C. Growth of each strain of microorganism was carried out under sterile conditions in stirred antibiotic flasks (100 ml final volume) that contained brain heart broth at 37 C. Stirring was carried out by a magnetic agitator (Variomag) at 400 rpm. The inoculum concentrations were obtained by serial dilution and were determined on Petri dishes filled with the same growth medium or Luria Bertani medium (LB broth containing, per liter: 10 g bactotryptone, 5 g bacto yeast extract, 5 g sodium chloride) with 1.5% agar added. Reactants and chemicals The KOH used was of reagent grade or higher (Prolabo, Paris, France) and was kept at 4 C between the experiments. Ampicillin (Sigma, St Louis, Mo.) was dissolved in sterile distilled water. Next, several dilutions of the antibiotic solution were prepared with sterile distilled water (1150 mg ml1). All other chemicals were of reagent grade or higher. Indirect conductance assays In the operating conditions, the impedance was fixed by the conductance term. The device consisted of a software system enabling data acquisition via a connected bus which was wired to the measuring cells. Each measuring cell was an individual impedancemeter, able to measure the conductance change in the KOH solution for a given sample. These cells were composed of a tube containing a KOH solution and two electrodes (Fig. 1). The conductance values were acquired every 15 min and permanently recorded. Each cell could be activated independently of the others. Experimental procedures: assays on antibiotic flasks Assays were carried out on antibiotic flasks of 125 ml, containing 100 ml of brain heart broth and with a head-space of 25 ml. The flasks were closed by a rubber septum, which was equipped with a Teflon layer on its bottom and an aluminum cap. One milliliter of inoculum was injected through the septum, using 12 ml syringes (Codan Luer) with sterile needles (0.840.0 mm; Terumo). The CO2 produced was carried to the measuring cell through 20 mm of Tygon 16 tubing (Masterflex; Cole Parmer, Chicago, Ill.) with internal and external diameters, respectively, of 3.1 mm and 3.2 mm. The flasks were inoculated with various strains of microorganism and then incubated at 37 C (in a Jouan incubator) and stirred at 400 rpm, if necessary. The stirring was carried out by a magnetic stirrer (length 15 mm) pulled by a magnetic agitator multipost (Variomag), with parameters fixed by a controller. Assays on French canned beans Assays were carried out on fine French canned beans (volume 850 ml, total net weight 800 g, dry net weight 440 g) from the same batch and sold in hypermarkets (cheapest product). The composition of this product was: fine French beans, water, salt. The cans were artificially inoculated by syringe injection of 1 ml of microorganism suspension. Assays on apple pure in glass jars

Materials and methods

Microorganisms The microbial strains tested were E. coli ATCC 25922, Bacillus subtilis ATCC 6051, Saccharomyces cerevisiae SAFE BREWT58 BBE022002BVB, S. cerevisiae ATCC 9763, and S. cerevisiae var. ellipsodus CIP 635.66. Bacteria and yeasts were stored at 80 C and 20 C, respectively, in 20% glycerol and then thawed and

Assays were carried out on apple pure in glass jars (volume 720 ml, net weight 710 g), from the same batch and sold in hypermarkets. The composition of this product was: apples 92%, sugar, glucose syrup, total content of sugar 18%. Again, 1 ml of microorganism suspension was injected by syringe into the glass jars.

37 Fig. 1 Scheme of the device. Elec. Electrodes

Results
Optimal device parameters First, the characteristics and parameters of the functioning of the device were determined, in order to optimize the conductance response. The dynamics of CO2 absorption and the ratio between the impedance variation and the amount of CO2 produced were investigated according to Dzenclos et al. (1994), using a RABIT direct impedancemeter. The conductance response was calculated by measuring the conductance values of the solutions of KOH and KHCO3, corresponding to the initial and final chemical species during the measuring (see Introduction). The conductance response was a function of the KOH concentration, the volume, and the electrode height. With KOH concentrations of 115 g l1, a KOH volume of 16 ml, and an electrode height of 1.0 4.5 cm, it was found that the optimal conditions were: KOH concentration 7 g l1, KOH volume 5 ml, electrode height 4.5 cm. These conditions offered a cell constant of 0.145 cm1, measured with 0.1 mol KCl l1. Temperature affects the conductance value; and the experimental temperature must therefore be fixed to avoid negative false detections (Firstenberg-Eden and Eden 1984). Our impedancemeter was slightly affected by temperature changes, i.e. about 70 S per degree Celsius, representing 0.079% of the baseline conductance value (see Sensitivity, below). The device recorded conductance variations, according to the quantity of CO2 produced by microorganisms. After calibration with known volumes of CO2, it appeared that the conductance variation was 12.9 S mol1.

Sensitivity Without any CO2 introduced, the baseline conductance was typically 89.20.13 mS, i.e. a variation of 0.15%. This was considered the lowest noiseless conductance variation, i.e. the sensitivity of the device. With a ratio of 12.9 S mol1, the detection sensitivity was 10.9 mmol of CO2. These results were obtained using a laboratory device with an 8-bit analog digital converter. The use of a 12- or 24-bit converter would obviously provide a higher sensitivity. Repeatability of the conductance responses Having confirmed the parameters and functions of the device, it was possible to test the conductance response of this device in microbiological applications. The bacterial strain E. coli ATCC 25922 was chosen as a model. In Fig. 2, it is remarkable to observe the almost completed superimposition of the three obtained curves. This is also true in the detail of all parts of the curves, as was previously shown by Eden and Eden (1984). In the first step of the apparent lag phase, the conductance value just reflected the ionic load initially present in the measuring cell, the KOH solution, without any detectable kinetics of bacterial growth and hence without any reaction between the KOH and the CO2 produced. This phase had a duration of 8.250.25 h for each sample, considering the inoculum conditions. At the end of this phase, the measured conductance of visible latency had an average of 89.20.13 mS. The slopes of the visible exponential phase are identical, with an average of 2.33 mS min1. The average conductance reached at the

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Fig. 2 Conductance curves of three suspensions (diamonds, squares, triangles, respectively) of Escherichia coli ATCC 25922 in brain heart broth (100 ml), at 2104 colony-forming units (CFU) ml1, 37 C, 400 rpm)

Fig. 3 Relation between the detection time and the initial E. coli ATCC 25922 load

end of the exponential phase was at 82.70.13 mS, with a variation coefficient equal to 0.16%. The detection time (DT) is determined when the conductance variation between consecutive records is superior to the standard deviation of the baseline in the apparent lag phase, which reflects a positive detection and contamination of the sample. The determination of DT is the result of the combination of two phenomena: microbial growth (the true lag phase) and gas transfer (gas transfer between liquid and gas phases, tubing length, kinetics between CO2 and KOH). Thus, a delay can appear between the exponential phase of growth and the determination of DT. Experimentally, we found a delay of 2.000.25 h for E. coli growth. In our experiments, the DT was 8.250.25 h, with a variation coefficient of 3%. Therefore, we conclude that it is possible to use this device for determining microbial activity by the DT, with a very good repeatability. Linearity of conductance responses The apparent lag phase is inversely proportional to the concentration of the tested inoculum. Plotting the logarithm of the inoculum concentration as a function of DT, a linear correlation was observed for 1108 colony-forming units (CFU) ml1 for eight suspensions of E. coli (Fig. 3) The same assays of repeatability and linearity were achieved with the following microbial types under the same conditions: B. subtilis ATCC 6051, S. cerevisiae SAFE BREWT58 BBE022002BVB (industrial strain), S. cerevisiae var. ellipsodus CIP 635.66, and S. cerevisiae ATCC 9763. They gave similar results in the range 1 108 CFU ml1 (data not shown). These results were obtained with pure cultures in selected media. Therefore, as our impedancemeter was conceived to detect microbial contamination in foodstuffs, we tested the response in typical foodstuff media. However, it was necessary first to determine to what extent the product affects the detection and thus the

profile of the conductance variation. A natural degassing appears for some products, notably for vegetable products. It is thus observed with products stemming from industrial production that the release of CO2 is intrinsic in the product and modifies the conductance of the KOH. We can wrongly consider the detection as positive, leading to a false positive. For example, French beans, potatoes, small peas, mushrooms, spinach, and convenience foods spontaneously release CO2 (Bombe, personal communication). Natural release of CO2 from foodstuff products These tests were made in our laboratory on artificially contaminated commercial products whereas the Centre Technique des Conserves et Produits Agricoles (CTCPA, Dury, France) performs routine assays in collaboration with foodstuff factories. Also, for the same product, variations are observed when products stem from different production units or from different manufacturers. For example, the percentage of natural CO2 release oscillates between 10% and 21% for potatoes. In our experiments, the control (KOH without CO2) showed a quasi-null conductance change, whereas the French beans showed a natural release of 6.6 mol l1 h1 and the inoculated canned food showed about 60 mol l1 h1. For the latter, the CO2 production rates were calculated by the slopes of the conductance changes after the DT. Detection assays on artificially microbiologically contaminated French canned beans Figure 4 shows the conductance changes in KOH solution due to the CO2 produced in canned food which had been artificially contaminated by E. coli ATCC 25922. The

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Fig. 4 Conductance curves by indirect impedance for three different suspensions of E. coli ATCC 25922 in French canned beans. Squares 2.35104 CFU ml1, triangles 2.35101 CFU ml1, crosses 2.35102 CFU ml1, diamonds control

Fig. 5 Conductance curves by indirect impedancemetry for three different suspensions of Saccharomyces cerevisiae SAFE BREWT58 BBE022002BVB in glass jars of apple pure. Squares 6.1103 CFU ml1, triangles 6.1 CFU ml1, crosses 6.1103 CFU ml1, diamonds control

changes were caused either by a natural CO2 release from the vegetable product or by CO2 produced during microbial growth. The conductance curves of the artificial contamination conditions present decreasing values of conductance and a DT can be determined. The higher the initial count, the shorter the DT. A microbial load was detected with an initial count of ten bacteria per can (volume 850 ml); and this detection was carried out within 30 h, indicating the sensitivity and rapidity of the detection achieved with this device. However, problems sometimes appeared when liquids moved towards the detection cell. Cans are filled with vegetables immersed in liquid; and the quantity of this liquid is such that it leaves a small head-space. When an important quantity of CO2 is produced, the pressure increases and the seal forces the cans to blow up. We recommend the use of transparent tubing to facilitate the surveillance of such phenomena. A detection test with artificial contamination by B. subtilis ATCC 6051 was likewise carried out on the same product; and the DT was 12.5 hours for 5.88102 CFU ml1. The CO2 production rate was 8.8 mol l1 h1 and the rate for the control was equal to 6.4 mol l1 h1. These results are slightly lower than those of E. coli for approximately the same load of microorganisms. The indirect impedance technique is faster than traditional tests, for which the duration time is equal to 7 days for can-incubation at 37 C. This is the coupled duration time/temperature of incubation normally used in European agrofood industries, adapted from the French standardized methods (AFNOR 1976, 1977) V 08-401 and V08-402. In addition, after detection, the identification step to be carried out takes 2472 h.

Detection assays on artificially microbiologically contaminated glass jars of apple pure Jars of apple pure were artificially contaminated with S. cerevisiae SAFE BREWT58 BBE022002BVB. Figure 5 shows the decreasing conductance curves for each condition, in comparison with the control (non-inoculated apple pure). The DTs are once again correlated to the initial load. In this experiment, the microorganism suspensions were injected into the mass of the product. Detection of a microbial load equal to a dozen yeast (in a volume of 720 ml) was carried out in less than 25 h, which underlines the rapidity of the device in comparison with traditional methods. This detection was faster than classic microbial techniques, e.g. a stability test lasting 7 days at 37 C, followed by an identification step carried out for 2472 h. The same assays were performed on apple pure with E. coli ATCC 25922 and B. subtilis ATCC 6051, but no detection of any bacterial load was recorded, even when microorganism suspensions were injected into the mass of the product. Either there was no growth in the samples or the viscosity of the product did not allowed the diffusion of any CO2 produced during microbial growth, i.e. the gas was entrapped in bubbles and could not diffuse to the surface. The same experiments, carried out as for both strains above, but with a deposit on the surface of the product, show that there was no growth at all. A very small growth on the surface (0.98 mol l1 h1) was found, but no DT could be determined because there was no significant break of slope, showing this product does not allow the growth of microorganisms like E. coli or B. subtilis. In order to optimize our protocol, the assay must be agitated to homogenize the product and microorganisms. The limits and drawbacks of the method thus clearly appear for products like jams, jellies, or syrups, but not for liquids or products with a low viscosity, like fruit

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Sawai et al. (2002) described the same kind of results, but with a modified Bactometer device, while our results were performed with a pre-commercial device. Our device and the indirect impedance technique in general can rapidly detect the presence of compounds in products, such as antibiotics in milk, or it can measure the sensitivity of a microorganism towards a given compound.

Discussion
Microbiological control is today mandatory for industry. It is a question of guaranteeing the quality of products, to be sure of their perfect harmlessness towards health and to maintain their trade quality throughout the distribution networks to the consumer. Traditional microbiological methods require laborious manipulations and especially they entail an important delay, because the growth of microorganisms requires time to become detectable in selective media. These constraints are less and less compatible with the performance of industrial production, which tends to adapt itself increasingly to the request of the commercial market. The beginning of the 1970s was characterized by a debate concerning the classic microbiological methods based on enumeration (CFU-counts). Based on the indirect impedance technique, our device perfectly integrates the criteria of alternative or rapid methods. Automation of this technique is reasonably easy to carry out. This method is based directly on the metabolic activity of microorganisms, more particularly on their faculty to grow on complex media. Indeed, the device is able to detect microbial growth independent of the medium, which confers on the method a great flexibility in use and a wide adaptability in the detection of microorganisms in the agrofood industry. As such, the indirect impedance method can be qualified as rapid and relevant. The device allies a sensitivity threshold of 10.9 mol of CO2, a rapid response (detecting 1108 microorganisms ml1 within 550 h), and an excellent repeatability (lower than 3% variation). As compared with the other available techniques for CO2 measurement (Table 1) our impedancemeter is not the most sensitive, but seems to

Fig. 6 Detection time for E. coli ATCC 25922 as a function of ampicillin concentration. White diamonds Detection time, black squares conductance variation)

juices (Deak and Beuchat 1993), milk (Ribeiro et al., publication in preparation), or vegetable products in cans, like e.g. French beans. Sensitivity tests: challenge tests Microbial growths can be affected by numerous chemical species; and the indirect impedancemeter can thus be used as a practical technique to detect the presence of inhibitors. As an example of the potential of our device, we tested the effect of an antibiotic, ampicillin, on the growth of E. coli ATCC 25922 (approximately 2.2101 CFU ml1) in brain heart broth. The conductance variation and the DT were reported as a function of the ampicillin concentration (Fig. 6). When the concentration was inferior to 10 mg ampicillin l1, no effect was observed, while for 50 mg l1 and 80 mg l1, the DT increased rapidly. For 100 mg l1 and 150 mg l1, no conductance variation and obviously no DT was measured. The results show that the addition of antibiotics to sensitive microorganisms increases the DT and reduces the electric response. Similar results were obtained in direct conductance (Firstenberg-Eden and Eden 1984; Huang et al. 1998; Chang et al. 2000). In the indirect conductance method,

Table 1 Comparison between rapid techniques for CO2 determination Technique Manometry Radiolabeling Sensitivity (mol CO2) 30 106 Specificity Low High Limits and drawbacks Fine temperature regulation Regulatory restrictions. No spontaneous generation. 14C is introduced into a metabolizable reactant High-cost technique Reference Wick et al. 2001 Friedlander 1981

Infrared

0.16

Medium

Indirect impedance

10.9

Medium

See text

Obtained by calculation (based on TOC analyzer documentation; OI Analytical, Texas) This study

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be the best compromise between sensitivity, technical constraints, and cost. Each measuring cell is an individual impedancemeter that could be activated independently of the others. These criteria provide an alternative method of choice against traditional methods of detection. Our impedancemeter device is able to detect microbial growth in foodstuff products, packaged in metal cans or in glass jars. The detection delay is less with the indirect impedance technique than with traditional methods. Indeed, this technique enables detection of contamination within a short time; and the DT is less than the 7 days which is the duration time applied in agrofood industries. At least two French AFNOR (2000, 2002) standards V08-105 and V08-106 give directives for setting-up an impedancemeter technique for quality control in food microbiology and especially the indirect technique for E. coli detection in live shellfish. Some assays are being prepared by the CTCPA to provide the first results concerning the use of this device in the field of stability control, in routine tests, and on industrial sites (canning factory, dairy industry, etc.). Its uses can be multiple, in routine tests (quality control of foodstuffs, pharmaceuticals, cosmetics, the environment), or in the laboratory (research and development). The total independence of media and microorganisms makes this device a very valuable global measuring system.
Acknowledgement We are very grateful to Christine Jespersen for careful reading of the manuscript.

References
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