Impedance microbiology

Impedance microbiology is a microbiological technique used to measure the microbial number density (mainly bacteria but also yeasts) of a sample by monitoring the electrical parameters of the growth medium. The ability of microbial metabolism to change the electrical conductivity of the growth medium was discovered by Stewart^[1] and further studied by other scientists such as Oker-Blom,^[2] Parson^[3] and Allison^[4] in the first half of 20th century. However, it was only in the late 1970s that, thanks to computer-controlled systems used to monitor impedance, the technique showed its full potential, as discussed in the works of Fistenberg-Eden & Eden,^[5] Ur & Brown^[6] and Cady.^[7]

Principle of operation

Figure 1: Equivalent electrical circuit to model a couple of electrodes in direct contact with a liquid medium

When a pair of electrodes are immersed in the growth medium, the system composed of electrodes and electrolyte can be modeled with the electrical circuit of Fig. 1, where R_m and C_m are the resistance and capacitance of the bulk medium, while R_i and C_i are the resistance and capacitance of the electrode-electrolyte interface.^[8] However, when frequency of the sinusoidal test signal applied to the electrodes is relatively low (lower than 1 MHz) the bulk capacitance C_m can be neglected and the system can be modeled with a simpler circuit consisting only of a resistance R_s and a capacitance C_s in series. The resistance R_s accounts for the electrical conductivity of the bulk medium while the capacitance C_s is due to the capacitive double-layer at the electrode-electrolyte interface.^[9] During the growth phase, bacterial metabolism transforms uncharged or weakly charged compounds of the bulk medium in highly charged compounds that change the electrical properties of the medium. This results in a decrease of resistance R_s and an increase of capacitance C_s.

In impedance microbiology technique works this way, the sample with the initial unknown bacterial concentration (C₀) is placed at a temperature favoring bacterial growth (in the range 37 to 42 °C if mesophilic microbial population is the target) and the electrical parameters R_s and C_s are measured at regular time intervals of few minutes by means of a couple of electrodes in direct contact with the sample.

Until the bacterial concentration is lower than a critical threshold C_TH the electrical parameters R_s and C_s remain essentially constant (at their baseline values). C_TH depends on various parameters such as electrode geometry, bacterial strain, chemical composition of the growth medium etc., but it is always in the range 10⁶ to 10⁷ cfu/ml.

When the bacterial concentration increases over C_TH, the electrical parameters deviate from their baseline values (generally in the case of bacteria there is a decrease of R_s and an increase of C_s, the opposite happens in the case of yeasts).

The time needed for the electrical parameters R_s and C_s to deviate from their baseline value is referred as Detect Time (DT) and is the parameter used to estimate the initial unknown bacterial concentration C₀.

Figure 2: R_s curve and bacterial concentration curve as function of time

In Fig. 2 a typical curve for R_s as well as the corresponding bacterial concentration are plotted vs. time. Fig. 3 shows typical R_s curves vs time for samples characterized by different bacterial concentration. Since DT is the time needed for the bacterial concentration to grow from the initial value C₀ to C_TH, highly contaminated samples are characterized by lower values of DT than samples with low bacterial concentration. Given C₁, C₂ and C₃ the bacterial concentration of three samples with C₁ > C₂ > C₃, it is DT₁ < DT₂ < DT₃. Data from literature show how DT is a linear function of the logarithm of C₀:^[10][11]

${\displaystyle DT=A\cdot \log _{10}(C_{0})+B}$

where the parameters A and B are dependent on the particular type of samples under test, the bacterial strains, the type of enriching medium used and so on. These parameters can be calculated by calibrating the system using a set of samples whose bacterial concentration is known and calculating the linear regression line that will be used to estimate the bacterial concentration from the measured DT.

Figure 3: R_s curves for samples featuring different bacterial concentration as function of time

Impedance microbiology has different advantages on the standard plate count technique to measure bacterial concentration. It is characterized by faster response time. In the case of mesophilic bacteria, the response time range from 2 – 3 hours for highly contaminated samples (10⁵ - 10⁶ cfu/ml) to over 10 hours for samples with very low bacterial concentration (less than 10 cfu/ml). As a comparison, for the same bacterial strains the Plate Count technique is characterized by response times from 48 to 72 hours.

Impedance microbiology is a method that can be easily automated and implemented as part of an industrial machine or realized as an embedded portable sensor, while plate count is a manual method that needs to be carried out in a laboratory by long trained personnel.

Instrumentation

Over the past decades different instruments (either laboratory built or commercially available) to measure bacterial concentration using impedance microbiology have been built. One of the best selling and well accepted instruments in the industry is the Bactometer^[12] by Biomerieux. The original instrument of 1984 features a multi-incubator system capable of monitoring up to 512 samples simultaneously with the ability to set 8 different incubation temperatures. Other instruments with performance comparable to the Bactometer are Malthus by Malthus Instruments Ltd (Bury, UK),^[13] RABIT by Don Whitley Scientific (Shipley, UK)^[14] and Bac Trac by Sy-Lab (Purkensdorf, Austria).^[15] A portable embedded system for microbial concentration measurement in liquid and semi-liquid media using impedance microbiology has been recently proposed.^[16][17] The system is composed of a thermoregulated incubation chamber where the sample under test is stored and a controller for thermoregulation and impedance measurements.

Applications

Impedance microbiology has been extensively used in the past decades to measure the concentration of bacteria and yeasts in different type of samples, mainly for quality assurance in the food industry. Some applications are, the determination of the shelf life of pasteurized milk^[18] and the measure of total bacterial concentration in raw-milk,^[19][20] frozen vegetables,^[21] grain products,^[22] meat products^[23] and beer.^[24][25] The technique has been also used in environmental monitoring to detect the coliform concentration in water samples as well as other bacterial pathogens like E.coli present in water bodies,^[26][27][28] in the pharmaceutical industry to test the efficiency of novel antibacterial agents^[29] and the testing of final products.

References