**3.3. Bacterial cell wall identification based on their dielectric properties**

The identification of bacterial strains in biological media is a matter of interest in very different fields of modern life. Examples are in food hygiene and food industry, catering and gastronomy [44], [45], in environmental research activities, fermentation processes for the production of medical drugs, such as insulin, antibiotics, and other [46]-[48], and in the diagnosis of infections in clinical and veterinarian applications [49]. Depending on the respective research and application field, bacterial strains are currently detected by complex methods, for example: polymerase chain reaction (technique to amplify a single or a few copies of a piece of DNA), fluorescent *in situ* hybridization, DNA microarray and Ramanspectroscopy, etc.

278 Ultra-Wideband Radio Technologies for Communications, Localization and Sensor Applications

) and liver, muscle or kidney ( 40

high-water-content tissues show similar permittivity

bovine origin can be distinguished (Fig. 20, right panel).

measuring points (left panel). Real and imaginary part of permittivity

[43].

permittivity ( 8

independent measurements [25].

their properties, biological macromolecules and fluid compartments inside and outside membranes [41]. Special topics include a summary of the significant advances in theories on counter ion polarization effects, dielectric properties of cancer *vs.* normal tissues, properties of low-water-content tissues [42], and macroscopic field-coupling considerations. The dielectric properties of tissues are often summarized as empirical correlations with tissue water contents in other compositional variables. The bulk electrical properties of tissues are needed for many bioengineering applications of electric fields or currents, and they provide insight into the basic mechanisms that govern the interaction of electric fields with tissue

Using devices with our own configurations, the dielectric properties of different porcine and bovine tissues were determined [25]. Different measuring points were defined on the surface of udder, fat, liver, muscle, and kidney of porcine and bovine tissue (homogenous structure) and the permittivity of these points was measured three times (selected tissues in Fig. 20, left panel). Afterwards, the tissue under these measuring points was excised and dried to calculate the water content. Water content and permittivity ε´ were related to each other, so we could clearly differentiate between fat, low-water-content tissue, with a low

**Figure 20.** Determination of the dielectric properties of different porcine and bovine tissues at defined

The identification of bacterial strains in biological media is a matter of interest in very different fields of modern life. Examples are in food hygiene and food industry, catering and gastronomy [44], [45], in environmental research activities, fermentation processes for the production of medical drugs, such as insulin, antibiotics, and other [46]-[48], and in the diagnosis of infections in clinical and veterinarian applications [49]. Depending on the

bovine tissue in relation to the water content (right panel). The standard error represents six

**3.3. Bacterial cell wall identification based on their dielectric properties** 

) as high-water-content tissues. The

values whereas fat of porcine and

at 2 GHz of porcine and

Different studies have shed some light into the biomass determination of different microbial suspensions *via* dielectric spectroscopy. Mishima *et al.* investigated growth kinetics of bacterial, yeast and animal cells by dielectric monitoring in the frequency range of 10 kHz - 10 MHz [50]. The determination of bacterial growth by dielectric measurements was also shown by Harris *et al.* [51]. Jonsson *et al.* measured the concentration of bacterial cells *via* indirect methods based on the dielectric determination of ions in the suspension, which are released by killed cells [52]. Benoit *et al.* showed that it is possible to discriminate the hydrophobic or hydrophilic features of bacterial suspensions by determining the dielectric permittivity [53]. Nevertheless, no data are available for discrimination on the basis of bacterial structures *per se*, such as the presence of Gram-positive or Gram-negative bacterial strains in biological samples [54].

Therefore, two different Gram-positive bacterial strains (*Micrococcus luteus* and *Bacillus subtilis*) and two Gram-negative bacterial strains (*Escherichia coli* and *Serratia marcescens*) were cultivated under standard conditions using Standard I media and shaking flasks. Bacterial strains were incubated for 24 h at 37°C in an incubation shaker. To assess whether the Gram-status of bacteria could be determined by dielectric spectroscopy, bacterial suspensions were transferred to 50 ml tubes and centrifuged. The supernatant (liquid above precipitate) was removed, the pellet was washed in 0.9% sodium chloride solution and, finally, the dielectric properties of the bacterial biomass (pellet of 10 ml) were determined. Dielectric spectroscopy of bacterial strains and suspensions was performed using a network analyzer in a frequency range from 30 kHz to 6 GHz (HP 8753D) and a coaxial probe (High temperature probe). The real and imaginary part of permittivity was determined in a frequency range from 50 MHz to 300 MHz [54].

In the frequency range between 50 and 300 MHz, dielectric spectroscopy revealed higher values of the real part of permittivity ( 160 Gram-positive) of the Gram-positive bacterial strains *Micrococcus luteus* and *Bacillus subtilis* compared to the Gram-negative strains *Escherichia coli* and *Serratia marcescens* ( 100 Gram-negative). From each strain the same cell count and volume was measured. Particularly at a frequency of 50 MHz

(maximum of discrimination), the real part of permittivity of both Gram-positive strains was about 60 units higher than of the Gram-negative strains (Fig. 21)

The clear discrimination between the Gram-positive strains *Micrococcus luteus* and *Bacillus subtilis* as well as the Gram-negative strains *Escherichia coli* and *Serratia marcescens* at a frequency up to 100 MHz can be attributed to the β-dispersion. At these frequencies, proteins and other macromolecules of the bacterial cells polarize according to Markx *et al*. [35]. This effect decreases at frequencies above 100 MHz. With increasing frequency the influence of water becomes more prominent.

ultraMEDIS – Ultra-Wideband Sensing in Medicine 281

(red dots in Fig.

and

All bacterial strains investigated in the present study revealed a characteristic time-

22). The growth kinetics was not influenced by the presence of accumulated metabolites in the culture medium since supernatants (liquid above precipitate) of every bacterial culture showed the same permittivity as the Standard I culture media (Fig. 22; 85 78

In therapeutic or diagnostic applications or biological effects of the electromagnetic field, dosimetric evaluations are greatly dependent on the precise knowledge of the dielectric parameters of biological tissues (relative permittivity ε and electrical conductivity σ). These parameters are sensitive to many influencing factors, which include the temperature of the target organ [55]. During radio-frequency or microwave radiation exposure, the internal temperature of tissue can change, thus influencing the electrical field distribution. For example, the evaluation of the lesion obtained by thermal ablation is a function of the relative permittivity and conductivity at 37°C and also of their evolution during heating. The influence of temperature in dielectric spectroscopy has been studied by several authors [56]-[58]. However, these effects remain misunderstood and the measured values are sparse

To find out in how far temperature-dependent changes in permittivity can result in a parameter identified by ultra-wideband technology, water and different tissues were examined. To assess the basic capability of UWB radar for monitoring local temperatures, dedicated phantom and *in vivo* experiments were performed. Dielectric spectroscopy of water at different temperatures (25 – 80°C in steps of 5°C) and corresponding experiments using porcine and bovine tissue, such as udder, liver, muscle, and kidney revealed a distinct decrease of permittivity with increasing temperature. Nevertheless, heating of tissues to more than 60 °C might also reduce permittivity due to the reduction of water content. No distinct organ-specific differences in the temperature-dependent dielectric properties have been found so far (Fig. 23). Only fat, as low-water-content tissue, exhibited no influence on

In addition to further studies with improved probes, corresponding analysis were performed using clinically approved temperature-based methods for tumor eradication, such as radio frequency ablation (RFA) or magnetic thermo ablation. For this experiment, a bovine liver was positioned onto a neutral electrode. The second, active electrode was launched into the liver tissue. Both electrodes together create a stress field, and the tissue around the active electrode becomes heated up to 60°C. Bi-static UWB antennas were first positioned in a distance to the region where RFA was thought to detect the signals of liver tissue itself. Then, the antennas were positioned above the region of radio frequency ablation, and changes in impulse response before, while and after radio frequency ablation were detected. The signal analysis displayed an increase of the impulse response during

dependent correlation between cell counts (black lines in Fig. 22) and

600 100 @ 50 300 MHz ) [54].

**3.4. Temperature influence on tissue permittivity** 

at various frequencies and exist only for some organs.

permittivity at different temperatures [59].

radio frequency ablation (data not shown) [59].

**Figure 21.** Discrimination of Gram-positive and –negative bacterial strains *via* dielectric spectroscopy. The diagram shows the real part of the permittivity of the biomass of Gram-positive bacterial strains (*Micrococcus luteus* and *Bacillus subtilis* [upper curves]) and Gram-negative bacterial strains (*Escherichia coli* and *Serratia marcescens* [lower curves]) in a frequency range between 50 and 300 MHz. The highlighted area shows the most obvious region of differentiation between Gram-positive and Gramnegative bacterial strains [54].

**Figure 22.** Monitoring of growth kinetics of four bacterial strains (growth phase). Red squares show the area under the curve (AUC) of the real part of permittivity in a frequency range between 50 MHz and 100 MHz derived from measurements during the bacterial growth phase. The permittivity of the cell suspension was taken hourly for 240 or 300 min. Black lines show the cell count per ml taken at the same time as permittivity was measured [54].

All bacterial strains investigated in the present study revealed a characteristic timedependent correlation between cell counts (black lines in Fig. 22) and (red dots in Fig. 22). The growth kinetics was not influenced by the presence of accumulated metabolites in the culture medium since supernatants (liquid above precipitate) of every bacterial culture showed the same permittivity as the Standard I culture media (Fig. 22; 85 78 and 600 100 @ 50 300 MHz ) [54].
