Non-linear dielectric spectroscopy of microbiological suspensions

Abstract

Background: Non-linear dielectric spectroscopy (NLDS) of microorganism was characterized by the generation of harmonics in the polarization current when a microorganism suspension was exposed to a sinusoidal electric field. The biological nonlinear response initially described was not well verified by other authors and the results were susceptible to ambiguous interpretation. In this paper NLDS was performed to yeast suspension in tripolar and tetrapolar configuration with a recently developed analyzer.

Methods: Tripolar analysis was carried out by applying sinusoidal voltages up to 1 V at the electrode interface. Tetrapolar analysis was carried on with sinusoidal field strengths from 0.1 V cm(-1) to 70 V cm(-1). Both analyses were performed within a frequency range from 1 Hz through 100 Hz. The harmonic amplitudes were Fourier-analyzed and expressed in dB. The third harmonic, as reported previously, was investigated. Statistical analysis (ANOVA) was used to test the effect of inhibitor an activator of the plasma membrane enzyme in the measured response.

Results: No significant non-linearities were observed in tetrapolar analysis, and no observable changes occurred when inhibitor and activator were added to the suspension. Statistical analysis confirmed these results.When a pure sinus voltage was applied to an electrode-yeast suspension interface, variations higher than 25 dB for the 3rd harmonic were observed. Variation higher than 20 dB in the 3rd harmonics has also been found when adding an inhibitor or activator of the membrane-bounded enzymes. These variations did not occur when the suspension was boiled.

Discussion: The lack of result in tetrapolar cells suggest that there is no, if any, harmonic generation in microbiological bulk suspension. The non-linear response observed was originated in the electrode-electrolyte interface. The frequency and voltage windows observed in previous tetrapolar analysis were repeated in the tripolar measurements, but maximum were not observed at the same values.

Conclusion: Contrary to previous assertions, no repeatable dielectric non-linearity was exhibited in the bulk suspensions tested under the field and frequency condition reported with this recently designed analyzer. Indeed, interface related harmonics were observed and monitored during biochemical stimuli. The changes were coherent with the expected biological response.

Figure 1

Non-linear dielectric spectrometer. (a) Equipment involved, (b) cylindrical flat electrodes tetrapolar cell, (c) low EEI impedance tetrapolar cell, and (d) tripolar cell. In (b) and (c) 1 and 4 correspond to outer electrodes, while 2 and 3 correspond to inner electrodes. In (d) 1 and 2 are connected together and the system acts as a tripolar analyzer, instead of a tetrapolar one. Numbers 1 to 4 correspond to the connections of the SI1287 unit depicted in (a).

Figure 2

Electric field distribution in the tetrapolar cells. Woodward's cell, (b) tetrapolar cell with flat electrodes, and (c) low EEI cell. All figures have been color-coded according to the scale showed in (d). For a better visualization, the field scale was clipped to a maximum of 2 V/cm. However higher values (~3.5 V/cm) were observed close to the outer electrodes in (a) and close to the acrylic corners in (c).

Figure 3

Difference between averaged measurements before and after addition of SMV in tetrapolar cells. The horizontal axes stand for the frequency and amplitude of the applied electric field. The vertical axis stands for the amplitude of the third harmonic measured at that combination of frequency and amplitude. The experiment was performed with the flat parallel electrodes cylindrical cell showed in fig. 1(b). Frequency range was 1 Hz~100 Hz (logarithmic scale, 11 steps), and field range was: (a) 0.167 V cm^-1 ~1.33 V cm^-1 (logarithmic scale, 11 steps). Surface is color-coded according to the color bar presented.

Figure 4

Third harmonic response measured in tripolar cell with gold electrode. Subsets (a) show the difference between a single measurement before and after a single injection of SMV. For the two peaks observed, the test, reference and difference spectrums were plotted at the right of the image. Subset (b) shows the spectra when the signal strength was 946 mV at 10 Hz. The difference measured for the third harmonic is 27.4 dB peak. Subset (c) shows the spectra when the signal strength was 376 mV at 6.3 Hz. The difference measured for the third harmonic is 26.3 dB. The surface in (a) is color-coded according to the color bar presented in fig. 2.

Figure 5

Third harmonic response measured in tripolar cell with stainless steel electrode. Subsets (a) show the difference between a single measurement before and after a single injection of SMV. For the valley observed, the test, reference and difference spectrums were plotted at the right of the image, in subset (b). The surface in (a) is color-coded according to the color bar presented in fig. 2.

Figure 6

Time evolution of the third harmonic when measured repeatedly on gold electrode adding SMV and glucose. (a) two different experiments where 1 mM of SMV was added at the time indicated by the arrow. (b) Effect of glucose on resting and sterilized yeast. Glucose (100 mM) was added at the timeindicated by the arrow in both experiments.