Appl Microbiol Biotechnol (1994) 42:232-238

Springer-Verlag 1994

T. D6zenclos M. Ascon-Cabrera D. Ascon J.-M. Lebeault - A. Pauss

Optimisation of the indirect impedancemetry technique; a handy technique for microbial growth measurement

Received: 1 December 1993/Received revision: 2 March 1994/Accepted: 11 March 1994

Abstract In the indirect impedancemetry technique, the CO2 produced during biological activity reacts with potassium hydroxide (KOH) solution, providing negative conductance variation. In this work, this technique was optimized, on a rapid automated bacterial impedance technique (RABIT) apparatus, developed by Don Whitley Sc. Ltd (UK). The K O H concentration and volume, as well as the temperature were tested, The dynamics of CO2 absorption and the ratio between the conductance variation and the amount of CO2 produced were examined. After injection of CO2 either directly in the K O H solution, or above the K O H solution, the best results were obtained with a K O H volume corresponding to immersion of the electrodes (0.7-1.2 ml), and with K O H concentrations of up to 7g/l, although 5-6g/1 is preferred. Decrease of 280 gS/gmol CO2 was obtained at 27C for a K O H concentration ranging from 0.5 to 8 g/1. All these results were slightly affected by temperature. However, it would be preferable for the CO2 produced to be bubbled directly into the K O H solution, in order to decrease the dynamic response of the system (gaseous transfer).

Introduction
Impedancemetry is now recognized as a practical and useful method of measuring microbial growth or activity. The first impedance microbiology experiment was achieved in 1899 by Stewart. He described the impedance variations during microbial metabolism on blood and serum, and suggested that bacterial growths could be monitored by electrical means.

T. D6zenclos - M. Ascon-Cabrera - D. Ascon J.-M. Lebeault A. Pauss ( ~ ) Division des Proc6d6s Biotechnologiques, Universit6 de Technologie de Compi~gne, B.P. 649, F-60206 Compi~gne cedex, France. FAX: 33-44 20 48 13

The impedance of a conducting materials such as biological medium can be simply defined as the resistance to flow of an alternating current as it passes through a conducting material. Considering the system as a series combination, the application of an alternating sinusoidal potential will produce a resultant current that is dependent on the impedance (Z) of the system, which in turn is a function of its conductance (G), capacitance (C) and applied frequency (F) as shown in Fig. 1 (Firstenberg-Eden and Eden !984). In biology, micro-organisms may produce ionized metabolites during growth; these metabolites will change the medium's conductivity. Figure 2 shows the relationship between the conductance variation, the ionic concentration and the bacterial concentration (Firstenberg-Eden and Zindulis 1984). The detection time (DT) corresponds to the point were the conductance variation rate is bigger than a predetermined value (e.g. 60 gS/min). The DT of the instrument (i.e. time for least variation of conductance and/or capacitance media) is thus correlated with the initial microbial content. The smaller the contamination, the longer the detection time. This phenomenon can be used to measure growth or bacterial activity. In practice, microbial growth or activity can be followed through conductance, capacitance, or impedance variations. The existing apparatus favours one or other of these parameters. The apparatus that is used in our laboratory is the rapid automated bacterial impedance technique (RABIT) developed by Don Whitley Sc. Ltd. (UK). It plots conductance as a function of time (Silley 1991b). The direct impedancemetry technique is used as a tool for quality control in the food (Easter and Gibson 1989) and dairy industries (Firstenberg-Eden 1986). It was utilized, for instance, for estimating contamination (Silverman and Munoz 1979; Noble et al. 1991), to determine the initial contamination of milk (Gnan and Luedecke 1982), or contamination after pasteurization (Bossuyt and Waes 1983), for a rapid

233

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However, the direct impedancemetry technique can not be used with all media, as they need to be optimized to obtain usable electric signal (neither too low or too high a salt concentration). Moreover, some microorganisms cannot be detected with this technique, because their growth does not induce a sufficient conductance change (due, for instance, to the formation of unionized products) (Owens and Wacher-Viveros 1986), or because they require selective growth media that not fulfil the ionic requirements. The problem could be overcome by using an indirect impedancemetry technique. This technique was first described by Owens et al. (1989); it has been developed for the RABIT. In this technique, the CO2-producing medium is placed on a small tube located above the electrodes and a K O H solution (Fig. 3). The acid-base reaction between CO2 and K O H leads to an important negative conductance change. This technique can be used for all CO2-producing micro-organisms, whatever their metabolism, and whatever the culture media (ionic content, viscosity, substrate nature, etc.). Rapid detection rates can be achieved for organisms such as Salmonella, Listeria, yeasts and moulds (less than 10 yeasts/g can be detected within 2 to 3 days, which is quicker than conventional techniques) (Silley f991a). The indirect impedancemetry technique has been perfectly adapted for the detection of Staphylococcus aureus, L. monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. (Bolton 1990, 1991). The detection of many vegetal pathogens (P. syringae, X anthomonas campestris, etc.) is possible with a short detection time (Franken and van der Zouwen 1993). This technique may be also used for measurements of the CO2 produced by Streptococcus thermophilus in pure and mixed culture with Lactobacillus (Ascon et al. unpublished data), or for population estimation of S. thermophilus (Ascon et al. 1993b). The degradation activity of adhered and suspended Pseudomonas cells cultured on 2,4,6-trichlorophenol was measured by the indirect impedancemetry technique (Ascon-Cabrera and Lebeault 1994).

Fig. 2 Relationship between bacterial growth, concentration of ions and bacterial concentration (Firstenberg and Zindulis 1984) (LAG end of lag period, DT detection time)

screening of milk microbial content (Cady et al. 1978), to determinate the potential shelf-life of pasteurized whole milk (Bishop et al. 1984), for the detection of some pathogens, that is some Enterobacteria (Cousins and Marlatt 1990) Lancefield group D cocci in skimmed milk powder (Neaves et al. 1988), Salmonella (Blackburn 1991; Easter and Gibson 1985), Clostridium botulinum (Gibson 1987), Listeria spp. (Phillips and Griffiths 1989), and for the kinetics and interactions of lactic acid bacteria (Ascon et al. 1993a).

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CO 2 absorbant medium

Fig. 3 Scheme of an indirect impedancemetry sample tube

234 In this work, we aimed to define the optimal experim e n t a l conditions for CO2 detection with a R A B I T system. We also focus on t h e relationship between the a b s o r b e d CO2 and the c o r r e s p o n d i n g conductance change, expressed in microsiemens per ~tmole of CO2.

Results
O p t i m a l K O H concentration Figure 4 shows a typical curve of the K O H conductance when excess CO2 was bubbled. The concentration was increased f r o m 1 g/l to 10 g/1 with 1 ml K O H solution. These first experimentations were carried out at the lower stable temperature, i.e. 27C. The initial c o n d u c t a n c e and the m a g n i t u d e change after CO2 addition increased linearly up to 8 g/1 (Fig. 5a). T h e y varied f r o m 4,200 pS to 32,700 pS, and f r o m 2,500 pS to 20,500 gS, respectively. A b o v e 8 g/1 the initial conductance of K O H solution was constant (32,700 BS). This value represented the m a x i m u m readable i m p e d a n c e by the RABIT. To establish the relationship between the conductance variation a n d the a b s o r b e d CO2, it was assumed that all K O H molecules had reacted with the excess C O > The a b s o r b e d CO2 was thus calculated via the a m o u n t of introduced K O H and the stoichiometric coefficient (1 m o l CO2 reacts with 2 mol K O H ) . Figure 5b showed that this coefficient was constant for a concentration ranging between 1 and 8g/1. I t was equal to 280 + 7 p S / g m o l CO2 at 27C. A b o v e 8 g/1 this coefficient was decreased. T h e very g o o d reproducibility of this technique m u s t be emphasized: all three p a r a m e t e r s obserged presented very s h a r p s t a n d a r d deviations.

Materials and methods

Reactants All reactants (hydrochloric acid, sodium carbonate, KOH), were of analytical grade (Prolabo, France). CO2 was of pure quality grade (Air Liquide, France). The KOH solutions were kept at 4C between the experiments.

Impedance measurements The conductances were monitored using a RABIT developed by Don Whitley Sc. Ltd. (UK). The apparatus can handle from 1 to 16 modules containing 32 electrode tubes that can each be analysed simultaneously. This is a completely multiworker system. The temperature range was up to 4C above ambient temperature up to 55 + 0.005C. The RABIT system may be used in the classical direct impedancemetry mode or the indirect impedancemetry mode.

Experimental procedures Indirect impedancemetry was calibrated and optimized through the monitoring of conductance change of the KOH solution with CO2 absorption into this solution. Conductance changes were recorded during 24 h 6-rain intervals. In practice, a KOH solution was placed in the electrode tubes. These tubes were tightly closed with rubber bungs, and placed in the RABIT incubator block. After stabilisation (1 h), a needle was pricked through the rubber bungs. Two comparative procedures were then followed: 1. Excess CO2 was bubbled into the KOH solution, and then converted into carbonate; the final quantity of COz absorbed was determined by the initial quantity of KOH (1 mol CO2 reacts with 2 mol OH-), 2. Known CO2 volumes were injected through the rubber bungs directly into the KOH solution. The KOH concentration (1 to 10 g/l), the KOH volume (0.3 to 5 ml), the test temperature (27-57C) and the corresponding dynamics were optimized. The initial conductance value (BS), the magnitude change (BS), and the relationship between the latter and the absorbed CO2 (gS/gmol), were measured and calculated.

O p t i m a l K O H volume The volume of a 6 g K O H / 1 solution was increased f r o m 0.3 up to 5 ml at 27C. The initial conductance and the m a g n i t u d e of change increased linearly with v o l u m e a u g m e n t a t i o n up to 1.2 ml. F r o m 1.5 ml those values were constant at 31,500gS and 20,000gS (Fig. 6a) respectively. F u r t h e r m o r e , the coefficient between the c o n d u c t a n c e change and the a b s o r b e d CO2 was m a x i m a l for a volume range of 0.7-1.2 ml (Fig. 6b).

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Microbial growth Growth of E. coli was performed under sterile conditions, in parallel in the impedancemetry tubes (3 ml as final volume) and in nonstirred erlenmeyer flasks (100 ml final volume) at 30C. The culture medium contained 10 g tryptone, 5 g meat extract, and 5 g sodium chloride per litre (Nutrient Broth, Biokar, France). The inoculum concentration was 4.105 colony-forming units (cfu)/ml, as determined by serial dilution on petri dishes filled with the same growth medium added with 1.5% agar.

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Fig. 4 Typical conductance variation for CO2 injection into a KOH solution

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Fig. 5 a Changes in the initial conductance (B) and in conductance change (A) as a function of the KOH concentration, when excess CO2 was bubbled into KOH (27C, 1 ml KOH). b Variation in the conductance change to absorbed CO2 ratio as a function of the KOH concentration, when excess COz was bubbled into KOH (27C, 1 ml KOH) This coefficient was Confirmed by injection of variable CO2 volume for 1.1ml of 6 g K O H / 1 at 37C (Fig. 7). The conductance change linearly increased with the CO2 volume, providing a coefficient of 257 pS/pmol. The difference between the previous coefficient (280 gS/pmol) and the latter is due to the different temperature and K O H volume used.

Fig. 6 a Changes in the initial conductance (,,) and in conductance change (a) as a function of the KOH volume, when excess COz was bubbled into KOH (27C, 6 g KOH/1). b Variation in the conductance change to absorbed CO2 ratio as a function of the KOH volume, when excessCO2 was bubbled into KOH (27C, 6 g KOH/1)
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T e m p e r a t u r e effect In 1 ml of a 6 g KOH/1 solution, our three parameters were m o n i t o r e d with bubbling excess CO2 in the range 27-47C. Figure 8a shows that the initial conductance was sensitive to temperature variation (about 250 ~tS/C), as well as the conductance change, and the conductance to COz coefficient (Fig. 8b). All three increased by a b o u t 1.5% per C.

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CO2 (gmol) Fig. 7 C o n d u c t a n c e v a r i a t i o n of 1.1 m l of a 6 g K O H / 1 s o l u t i o n for

increasing amount of gaseous CO2 volume ting with the K O H solution. T o measure this gas transfer, different experimental methods were pursued. They were c o m p a r e d to the usual bubbling of excess CO2 into K O H solution (6 g/l). Firstly 1 ml of 0.268 mmol carbonate/1 was set in the inner tube. Excess hydrochloric acid was added once, to create sudden CO2 production. Secondly, excess g a s e o u s CO2 was injected through the needle above the K O H solution Thirdly, k n o w n CO2 volumes were injected through the rubber bungs directly into the K O H solution.

Dynamic response of the conductance variations As indicated by Fig. 3, the COg-producing solution is usually located in a small tube placed above the electrodes. The COg produced appears in the gas phase, which must displace the initial gas phase before reac-

236
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The results of Fig. 9 demonstrate that the CO2 mass transfer was not instantaneous. About 25 _+ 3 and 245 _+ 16 min were needed to obtain, respectively, 50% and 95% of the total conductance change when the produced CO2 does not directly bubble into the K O H solution (at 27C with 6 g KOH/1), However, when a relatively large flux of CO2 arrived above the K O H solution, these delays were less important, i.e. 12 + 4 and 24 min for, respectively, 50% and 95%. When CO2 arrived directly into the K O H solution, the reaction was immediate, that is less than the least interval of 'time recording.

impedancemetry technique (continuous at 540 n m (circles and dotted line)

Fig. 10 Escherichia coli growth curve measured with the indirect line), and by optical density

dispersion of the experimental values along the growth curve, in contrast to the conductance values, which were automatically recorded.

Discussion

Growth measurement As an example, E. coli growth was performed at 30C in parallel in the impedancemetry tubes and in non-stirred erlenmeyer flasks. Growth was followed by optical density at 540 nm and by conductance change. An excellent correlation was found, as illustrated in Fig. 10. The total conductance change corresponds to a CO2 production of 33.3 gmol, using 305 S/mol as coefficient. It is emphasized that the usual optical density measurements were more time-consuming, but are also more subject to experimental errors, as shown by the

The indirect impedancemetry technique can be used for any CO2-producing medium. With the RABIT system, the optimal conditions of this technique were determined as follows: a K O H concentration of 6-7 g/1 and a K O H volume of 0.7-1.2 ml. The K O H concentration is limited by the electric data of the RABIT. A 32,768 pS corresponds to a resistance of 30.5 f~, the maximal readable value by the RABIT. The K O H volume corresponds to the volume defined by the electrode height. For K O H volumes greater than 1.2 ml, the "excedent" K O H is not "'seen" by the electrode. An effective maximal volume is thus defined by the inner tube diameter and the electrode height. The optimal operating conditions using RABIT are summarized in Table 1. As shown in the previous figures, these values are highly i:eproducible. On the other hand, within the

237 Table 1 Operating conditions for indirect impedance measurements with the rapid automated bacterial impedance technique (RABIT) under optimal conditions (1 ml of g KOH/1) Temperature (C) Parameters Initial conductance Conductance change Conductance change to absorbed CO2 (mS) (mS) (S/mol) 27 24.50 15.00 280 30 25.25 16.35 305 37 26.05 17.15 320 40 27.05 17.15 320 47 31.20 " 19.60 367

Table 2 Caculated readable C O 2 production by the RABIT and estimated CO2 production by different biological reactions. The estimations were based on the following hypothesis: (i) 3 ml reaction vo!ume; (ii) stoichiometric reactions and complete transformations of ~he substrates into CO2, the other products and micro-organisms (gt'owth yield of, respetively, 0.5, 0.1, and 0.1 for the aerobic culture and the ethanolic and methanogenic fermentations), within the usual

temperature range of these three fermentations; (iii) for the aerobic fermentation, the CO2 is exclusively derived from the dissolved O2, i.e. 8.25, 8.08, 7.81, 7.00, mg/1, respectively, at 25, 27, 30, 37C; (iv) in the ethanolic fermentation, 10 g/1 of glucose are transformed into ethanol, CO2 and yeasts; (v) 3 g/1 of acetate are transformed into methane and CO2 during the methanogenic fermentation Temperatur e (C)

Parameters Minimum readable CO 2 production Maximum readable GO2 production Magnitude order of CO2 production of: aerobic culture ehanolic fermentation methanogenic fermentation (gmol) (gl) (gmol) (gl) (I.tmol) (btmol) (gmol)

25 0:036 0.89 53.8 1315 0.39 300

27 0.036 0.89 53.6 1320 0.38 300

30 0.033 0.82 53.6 1330 0.37 300 135

37 0.031 0.79 53.6 1365 0.33 135

47 0.028 0.74 53.4 1400

27-47C range, they are slightly affected by temperature, about a 1.5% variation for a IC increase. This indicates that CO2 production can be directly quantified by the conductance change of the CO2 absorbent s0lution. Assuming, first, that the conductance parameters are the values reported in Table 1, and secondly that the minimum readable conductance change by the RABIT is about 10 btS, then the maximum and least absorbable CO2 can be calculated for different temperatures (Table 2). In Table 2, these results are compared to three theoretical estimated COg productions of different case-study biological media. The details are given in the table legend. ~According to Table 2, and with the present capacities, the RABIT can be successfully used to measured aerobic growth or activity. Conversely, the potential CO2 production of anaerobic fermentations is too great for its capacity. However, e'en:in those fermentations the RABIT can be used to handle the dynamics of methanogenic fermentation. For instance, it can accurately follow CO2 production until the saturation of K O H (i.e. 40% of the total potential production). The CO2 dynamic transfer in the tube was shown very important in this study. The classical design of the indirect impedancemetry technique (as indicated in Fig. 2) can induce a perverse effect. The results shown in: Fig. 9 demonstrate that the CO2 mass transfer :Would not be instantaneous, and that the dynamics of

fast bacterial C O 2 production can be masked by this mass transfer. The E. coli growth curve shows that this method can be actually used for growth measurement. Other growth examples measured with this technique Can be found in Ascon-Cabrera and Lebeault (1994), Ascon et al. (1993b), Franken and van der Zouwen (1993) and Owens et, al. (1989).
Acknowledgement The authors greatly acknowledge Mr J. P. Duval (from AES company, French dealer of the RABIT) for his precious help and advice.

References
Ascon-Cabrera M, Lebeault JM (1994) Degradation activity of adhered and suspended Pseudomonas cells cultured on 2,4,6trichlorophenol, measured by indirect impedancemen(y. Appl Environ Microbiol in press Ascon D, Cochet N, Lebeault JM (1993a) Kinetics and interactions of lactic acid bacteria measured by impedancemetry. In: (eds) VIth European Congress on Biotechn01ogy, Florence, Italy, 13-17 June 1993. 1, MO223 Ascon D, Cochet N, Lebeault JM (1993b) Population estimation of Streptococcus thermophilus by indirect impedancemetry. In: (eds) Forum or Applied Biotechnology, Gent (Belgium), 30 September-01 .October 1993 (in press) Bishop JR,.White CH, Firstenberg-Eden R (1984) Rapid impedimetric method for determining the potential shelf-life of pasteurized whole milk. J Food Prot 47:471-475

238 Blackburn C (1991) Detection of Salmonella in foods using impedance. Eu Food Drink Rev Winter: 35-40 Bolton FJ (1990) An investigation of indirect conductimetry for detection of some food-borne bacteria. J Appl Bacteriol 69:655-661. Bolton FJ (1991) Conductance and impedance methods for detecting pathogens. In: Vaheri A, Tilton RC, Balows A (eds) Rapid methods and automation in microbiology and immunology. Springer-Verlag, Berlin, pp 176-181 Bossuyt RG, Waes GM (1983) Impedance measurements to detect post-pasteurization contamination of pasteurized milk. J Food Prot 46:622-624 Cady P, Hardy D, Martins S, Dufour SW, Kraeger SJ (1978) Auto. mated impedance measurements for rapid screening of milk microbial content. J Food Prot 4l:277-283 Cousins DL, Marlatt F (1990) An evaluation of a conductanace method for the enumeration of Enterobacteriaceae in milk. J Food Prot 53:568-570 Easter MC, Gibson DM (1985) Rapid and automated detection of Salmonella by electrical measurements. J Hyg 94:245-262 Easter MC, Gibson DM (1989) Detection of microorganisms by electrical measurements. Rapid methods in food microbiology. Prog Ind Microbiol 26:57-100 Firstenberg-Eden R, Eden G (1984) Impedance microbiology. Wiley, New York Firstenberg-EdenR (1986) Food-borne microorganisms and their toxins. In: Pierson MD, Stern NJ (eds) Developing methodology. Dekker, New York, pp 129-144 Firstenberg-Eden R, Zindulis J (1984) Electrochemical changes in media due to microbial growth. J Microbiol Methods 2:103-115 Franken AAJM, Zouwen PS van der (1993) Direct and indirect conductimetry for identification and detection of plant patho~genic bacteria. J Appl Bacteriol 74:234-242 Gibson AM (1987) Use of conductance measurements to detect growth of Clostridium botulinum in a selective medium. Lett Appl Microbiol 5:19-21 Gnan S, Luedecke LO (1982) Impedance measurement in raw milk as an alternative to the standard plate count. J Food Prot 45:4-7 Neaves P, Waddell M J, Prentice GA (1988) A medium for the detection of Lancefield Group D cocci in skimmed milk powder by measurement of conductivity changes. J Appl Bacteriol 65:437-448 Noble PA(Ashton E, Davidson CA, Albritton WL (1991) Heterotrophic'plate counts of surface water samples by using impedance methods. Appl Environ Mierobiol 57:3287"3291 Owens JD; Wacher-Viveros MC (1986) Selection of pH buffers for use in conductimetric microbiological assays. J Appl Baeteriol 60:395-400 Owens JD, Thomas DS, Thompson PS, Timmerman JW (1989) Indirect conductimetry: a novel approach to the conductimetric enumeration of microbial populations. Lett Appl Microbiol 9:245-249 Phillips JD, Griftiths MW (1989) An electrical method for detecting Listeria spp. Lett Appl Microbiol 9:129-132 Silley P (1991a) Rapid automated bacterial impedance technique (RABIT). Soc Gen Microbiol Quarter 18:48-52 Silley P (1991b) "RABIT" in the food industry. Nutr Food Sci 131:2-3 Silverman MP, Munoz EF (1979) Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants. Appl Environ Microbiol 37:521-526 Stewart GN (1899) The changes produced by the growth of bactria in the molecular concentration and electrical conductivity of culture media. J Exp Med 4:235-243