Impedance can be simply defined as the resistance to flow of an alternating current as it passes through a conducting material. For a detailed review of impedance theory, see the publications by Eden and Eden (1984), Kell and Davey (1990), and Blake- Coleman (1993). When an electrolyte solution is exposed to an electrical field, the cations will move toward the negative cathode and the anions toward the positive anode. The movement of ions constitutes current flow within the solution, each ion carrying a fraction of the current in proportion to its degree of mobility and concentration. Conversely capacitance stores energy in an electrical field; a typical capacitor will consist of two conducting surfaces separated by a dielectric material. If a DC voltage is applied to a capacitor, current flow is prevented, but an AC voltage will produce current flow proportional to the rate of voltage change.
It is sufficient for our purposes to consider, as first proposed by Warburg (1899, 1901), that when two metal electrodes are immersed in a conductive medium the test system behaves either as a resistor and capacitor in series or as a conductor and capacitor in parallel. In the first case, the impedance of the system is measured as the dependence of the voltage on the current, the system being considered connected to a current of infinite resistance. In the second case, the system is considered connected to a voltage source of zero resistance, and the admittance describes the dependence of the current upon the voltage (Kell and Davey 1990)
In the case where the system is treated as a series combination, application of an alternating sinusoidal potential will produce a current that is dependent on the impedance (Z) of the system, which in turn is a function of its resistance (R), capacitance (C), and applied frequency (F):
Any increase in conductance, defined as the reciprocal of resistance, results in a decrease of impedance and an increase in current. The AC equivalent of conductance is admittance, defined as the reciprocal of the impedance. The units of impedance measurement are Siemens (S). Microbial metabolism usually results in an increase in conductance and capacitance; thus as microorganisms grow the impedance signal actually decreases. It therefore is preferable to follow the conductance and admittance signals, thereby positively reflecting the increase in metabolic activity arising from microbial growth.
It is clear that impedance is a complex term that comprises capacitative and conductive components, both of which are dependent on the frequency of the alternating current applied to the electrode system. At low frequencies the impedance reading is largely affected by the capacitance component, whereas at high frequencies it is predominantly affected by conductance.
Direct Impedance
It can be readily appreciated that changes in impedance of the growth medium result directly from the changes taking place in the bulk electrolyte. Substrates in microbiological growth media are generally uncharged or weakly charged but are transformed into highly charged end products as organisms follow normal metabolic pathways, thus increasing the conductivity of the test medium. Simple examples include the conversion of glucose from a non-ionized substrate to two molecules of lactic acid and a corresponding increase in conductivity. Further metabolism will take the lactic acid and three oxygen molecules to carbonic acid; the resulting three ion pairs include the smaller, more mobile bicarbonate ion, which is a more effective electrical conductor than the lactate ion. Hydrogen ions are nearly seven times more effective conductors than sodium ions (Eden and Eden 1984); therefore, one might predict that a weakly buffered medium would allow a greater impedance change than a more strongly buffered medium. For a more detailed appraisal of the effect of medium buffers on conductance, see the work of Owens (1985). It is important to stress that the principles of medium design, fundamental to traditional microbiology, are equally if not more important in impedance microbiology. First, a medium must be chosen that
will support and select for the growth of the test organism. Second, that medium needs to be optimized for an electrical signal. This is well illustrated by Staphylococcus aureus, which will grow in nutrient broth but does not produce a significant electrical response, whereas in Whitley Impedance Broth® (Don Whitley Scientific Ltd., United Kingdom), not only does it
grow well but it also produces a strong impedance signal. The growth of some organisms, particularly yeasts and molds, does not result in large changes in impedance. This is considered to be due in part to the fact that they do not produce strongly ionized metabolites, but rather non-ionized end products such as ethanol. Moreover, Suomalainen and Oura (1971) have shown that yeasts can absorb ions from solution, resulting in a net decrease in medium conductivity.
An impedance system can therefore be considered simply as measuring net changes in impedance in the culture medium at regular intervals. When a test is initially set up, the user defines the test criteria; when the rate of change of impedance exceeds this predetermined value, the system will detect growth. The time to detection (TTD) is a function of the size of the initial microbial population, the growth kinetics of the test organism, and the properties of the test medium. For a given test protocol, the TTD is inversely proportional to the initial microbial loading of the sample. At the point of detection, it is generally considered that there will be approximately 105 to 106 CFU/mL of the test organism present in the system. This will
vary depending on organism type and medium, but will be constant for any organism growing under defined test conditions. It is important to differentiate between this detection threshold and the sensitivity of an impedance system capable of detecting the presence of organisms at levels as low as <10 CFU/mL, providing the organisms are viable. It is well established that the electrode construction, stainless steel compared to platinum, will affect sensitivity of the test system. Eden and Eden (1984) showed that electrodes located at the bottom of a test cell resulted in detection thresholds 1 log cycle lower than with the same electrodes located at the top of the test cell.
The fact that real-time microbial activity is being measured rather than the activity at a single point in time is a significant and powerful feature, one that enables the system to detect the presence of low numbers of organisms. Several factors will affect time to detection. TTD will correlate only with the initial concentration of test organisms, providing that the generation time of the test population is more or less constant under the experimental conditions. Therefore, not only does incubation temperature need to be kept constant because of physico-electrical properties, as discussed earlier, but also because it will have a direct effect on the generation time of microorganisms.
Indirect Impedance
High salt concentrations are routinely used in many selective media and may be present in some test samples. For example, LiCI is incorporated into Baird-Parker staphylococcal medium at 5 g/L and into Oxford Listeria medium at 15 g/L. Also MgCl2
(36 g/L) is used in Rappaport-Vassiliadis broth for Salmonella isolation. The resultant high-impedance readings of these media are outside the normal working range of the direct impedance technique. However, using the indirect technique, the researcher can overcome these problems by monitoring micro bial metabolism via the production of carbon dioxide. In this instance potassium hydroxide is added to the impedance cell to bridge the electrodes. The inoculated culture medium is in a separate chamber and not in contact with the electrodes or potassium hydroxide. The unit is tightly sealed, so that any carbon dioxide produced as a result of normal metabolism is absorbed by the potassium hydroxide, causing a resultant decrease in impedance.
Principle of Detection by Indirect Impedance
The detection of carbon dioxide by indirect impedance was first described by Owens et al. (1989). Carbon dioxide produced as a consequence of microbial metabolism will dissolve in the aqueous growth medium until the solubility product is exceeded and the volatile gas diffuses into the headspace. In routine use the electrodes of the test measuring cell are bridged by a potassium or sodium hydroxide plug containing agar; alternatively, a simple solution may be used. As the CO2 is evolved, it
reacts with the alkaline solution, forming a carbonate with a reduced conductivity reading. This can be explained by the following reaction:
The pH of the test system is important, because the amount of CO2 in solution is governed by the Henderson-Hasselbach equation
that relates to the ionization constant of the carbonate formed in the culture, the pH, and the relative amount of volatile CO2
available.
A key factor in the design of the systems is that the measuring electrodes are separated from that part of the cell containing the growth medium. The indirect impedance method has several advantages relative to the direct conductance method. Indirect impedance allows the use of conventional media formulations, which thus need not be optimized to produce an optimal impedance response. Media containing high salt concentrations that are outside the measuring range of the direct method can be used. It also allows detection of some microorganisms that produce small or no detectable impedance changes by the direct IMPEDANCE 69
method. Indirect impedance can also be used with sample types that may physically interfere with the electrodes in the direct method.
The dynamics of carbon dioxide absorption and the ratio between the impedance variation and the amount of carbon dioxide produced have been investigated (Dezenclos et al. 1994). After carbon dioxide was injected either directly into the potassium hydroxide solution or above it, optimal results were obtained with potassium hydroxide (5–6 g/L) in a volume of 0. 7–1.2 mL, Impedance changes of 280 µ S/µ mol carbon dioxide were obtained at 27°C with potassium hydroxide concentrations of 0.5–8 g/L. This agrees well with the result predicted by Owens (−278.6 S.cm2.mol−1 carbon dioxide
absorbed; Owens et al. 1989). Not surprisingly the results were temperature dependent. While Owens et al. used aqueous solutions of 0.03 and 0.04 M KOH, Bolton (1990) used a semisolid 0.04 M KOH, and Druggan et al. (1993) used a 0.06 M semisolid KOH preparation. It is advisable that these alkaline solutions be degassed before use.
The work of Bolton (1990) has shown the indirect technique to be a powerful tool for working with strains of S. aureus, Listeria
monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, and Salmonella spp. With this method, the medium does not necessarily need to be optimized for electrical response, allowing media previously considered unsuitable to be used for impedance applications. Applications of indirect impedance technology also have considerable potential for workers who require a rapid, easily manageable, highly sensitive system for monitoring and quantifying CO2 production, whether in whole cell or isolated enzyme studies.