Influence of Cytoplasm Electrolyte Concentration on Maxwell− Wagner

The MW polarizability is studied in the present work in the range from 20 kHz to 20 MHz by change in the inner (cytoplasm) electrolyte concentration. ...
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J. Phys. Chem. B 2009, 113, 8375–8382

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Influence of Cytoplasm Electrolyte Concentration on Maxwell-Wagner Polarizability of Bacteria E. coli Alexandar M. Zhivkov* and Anna Y. Gyurova Institute of Physical Chemistry, Bulgarian Academy of Sciences, Acad. G. BoncheV Street, bl. 11, Sofia 1113, Bulgaria ReceiVed: NoVember 13, 2008; ReVised Manuscript ReceiVed: February 4, 2009

The electric polarizability of bacteria is considered in the literature to have a surface charge dependent (ChD) and a Maxwell-Wagner (MW) mechanism. We distinguish experimentally both the types of interface polarizability by the frequency of the electric field and the medium electrolyte concentration. It was shown in a previous work (Zhivkov, A. M.; Gyurova, A. Y. Colloids Surf., B 2008, 66, 201.) that the ChD component is shown up on the outer bacteria surface even at megahertz frequencies. The MW polarizability is studied in the present work in the range from 20 kHz to 20 MHz by change in the inner (cytoplasm) electrolyte concentration. The ion transport through the cytoplasmic membrane of alive and fixed by formaldehyde E. coli K12 is accelerated by adding of ethanol in low concentration. The frequency dependence and the kinetics of the electric polarizability and the size of the bacteria are investigated by conservative electric dichroism, based on the alteration of the optical density at orientation of the cells in electric field. The conclusion is that the internal MW component has the main contribution to the change in the total bacteria polarizability, as well as the external MW and the internal ChD components are not shown up. 1. Introduction The electric polarizability of colloid particles has two main contributions: surface and volume. As a rule, the last type is negligible in a suspension of particles with micrometer and submicrometer dimensions because of the big ratio between the total surface and volume of the particles. Two basic kinds of surface polarizability are known: Maxwell-Wagner (MW) and surface charge dependent (ChD) polarizabilities. The MW polarizability is defined as accumulation of electric charge (under the action of applied electric field) at the interface between two mediums having different volume electric conductivity and dielectric permittivity.1 Its relaxation takes place in the megahertz range and does not depend on the particle’s size. In the case of biological cells2,3 this polarization type is due to accumulation of ions in the cytoplasm nearby the bilayer lipid membrane, which is practically nonconductive. So, the value of the MW polarizability increases with the increase in the ionic strength of the electrolyte inside the bacteria cells. The ChD polarizability is defined as polarization of the electric double layer (EDL) of the particle,4,5 mainly of its diffuse part,6 and it is due to a migration of ions parallel to the surface of the particle. That is why the value of the ChD polarizability depends on the long size of the particles, the surface charge density, and the thickness of the EDL (determined by the ionic strength of the medium). The relaxation time of this kind of polarization depends on the square of the length of the particles7 and consequently may be observed at relatively low frequencies for cells with micrometer dimensions. In the case of charged solid particles the ChD polarizability plays the main role in their orientation in an electric field, which is proved by the dependence of the electro-optical effect (EOE) on the pH and the ionic strength of the medium: the polarizability practically disappears at the isoelectric point and/or at high electrolyte concentration.1,7 However, in the case of bacteria * Corresponding author.

the MW polarization should have the most significant contribution, because the conductive cell volume is enclosed by a nonconductive lipid membrane. The electric properties of bacteria, especially of E. coli, have beenwidelystudiedbyelectrophoresis,3,8 dielectricspectroscopy,9,10 and electro-optics.7,11,12 The electrophoretic mobility depends on the surface charge density only on the external side of the bacteria, whereas the dielectric spectroscopy and the electrooptics give information about all charges in the cells. The electro-optical methods are based on orientation of colloid particles in an electric field; the degree of orientation is proportional to the electric polarizability.7 Because the electrooptical methods are based on the alteration of the optical properties of the suspension, they do not require high particle concentration as the dielectric measurements do. This advantage of the electro-optics is emphasized in the electric light scattering and the conservative dichroism; the last method is based on the change of the optical density because of the light scattering in all directions.5,7 Usually in the electro-optical literature bacteria are treated like homogeneous solid particles, and therefore only the ChD polarizability is taken into account.5,7,11,12 However, the bacteria have more complex structure: liquid conductive cytoplasm and a solid nonconductive cover (cytoplasm membrane and cell wall). In particular, the cover of E. coli as a Gram-negative bacterium is built by two phospholipid membranes (outer and cytoplasmic) and a peptidoglycan monolayer between them.13 Because of the existence of two surfaces of the cell cover, external and internal ones, two ChD polarizabilitiessexternal (eChD) and internal (iChD) onessand two MW polarizabilitiessexternal (eMW) and internal (iMW) onessmust be considered. An alternative approach is the so-called electrophysical (EPh) model of bacteria, based on the MW type polarization.3 In that case the bacterium is considered like a homogeneous ellipsoid with complex dielectric permittivity. The values of the electric

10.1021/jp810020p CCC: $40.75  2009 American Chemical Society Published on Web 05/27/2009

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conductivity and dielectric permittivity are frequency-dependent and have characteristic frequencies for R- and β-dispersions.14 The EPh model in its more advanced variant considers the bacterial cell as a hollow ellipsoid having one or more layers with specific conductivity and permittivity of the core and each of the layers, respectively.3 The EPh model shows a presence of few dispersion regions with different relaxation frequencies and polarizability values along and across the bacterium.3 By adjustment of the parameters of the model it is possible to fit the theoretical frequency dependence of the polarizability to the experimental one. The main disadvantage of this approach is that the EPh model has too many parameters, which gives practically unlimited possibilities to fit the theoretical results to the experimental ones, and consequently it is not clear if the characteristics obtained in such a way are real. Beside that, the value of polarizability does not depend on the polarization of the EDL although its presence is taken into account by R-dispersion. We apply an experimental approach to distinguish the different types of surface polarization by changing the factors that influence the ChD and the MW polarizabilities. Our approach reduces to investigation of the dependence of the electric polarizability on the bacteria size and the ionic concentration (inside or outside of the cells) using the electric turbidimetry (known also as a conservative electric dichroism). In our previous work15 we have found out that the electric polarizability decreases with the increase in the ionic concentration of the outer medium (at a constant one inside) in the frequency range from 20 kHz to 2–5 MHz. Our study has detected the eChD polarizability even at these high frequencies, where the ChD component should disappear according to the dielectric and the electro-optical literature. In the present work we distinguish the iMW polarizability from the other components by changing the inner electrolyte concentration at almost the constant one in the outer medium. To this purpose we use ethanol, which increases the ionic permeability of the cell membrane16,17 and gives a possibility to investigate the frequency dependence and the kinetics of the polarizability at different inner electrolyte conductivities. Besides, we investigate the frequency dependence in the wide range from 20 kHz to 20 MHz, where the ChD polarizability decreases with the increase in the frequency to complete disappearance. 2. Materials and Methods 2.1. Materials. Bacteria culture of E. coli K12 was cultivated in standard Luria-Bertani broth supplemented with 1% glucose for 7.5 h at temperature of 37 °C and pH 7.0. The bacteria samples were taken from the medium, washed with distilled water on a 0.8 µm Millipore filter and suspended in aqueous medium with electric conductivity of 5 × 10-4 S/m exactly before the electro-optical measurements in the case of alive bacterial cells. To obtain a suspension of fixed (dead) E. coli K12, after the filtration bacteria were suspended in a fresh 3% aqueous solution of formaldehyde; the suspension was kept at 4 °C. The cells were washed from the formaldehyde and suspended in distilled water just before the beginning of the electro-optical measurements. The experiments connected to increase in the cytoplasm ionic concentration (Figures 4-7) were carried out as follows: The bacterial cells in two suspensions were incubated for 24 h in 150 mM KCl in absence and in presence of 5% ethanol, respectively. After that the bacteria were washed and suspended in distilled water just before the beginning of the electro-optical measurements.

Zhivkov and Gyurova The concentration of bacteria suspensions was adjusted to an optical density of 0.1 at 670 nm and 1 cm optical length in all the experiments. 2.2. Electric Dichroism. When an electric field is applied to the bacterial suspension, the optical density A is changed due to the orientation of the particles. The average degree of orientation of the particles is proportional to the torque M ) d · E, averaged on all the orientations, which is determined by the induced dipole moment d and the effective strength E of the electric field. The value of d ) γE is a linear function of E and the electric polarizability γ at not too high values of E. The absolute value of the EOE is defined as ∆A ) AE - A0, where the indexes E and 0 denote the applied electric field and its absence, respectively. The quantity of ∆A is determined by the mean degree of orientation F(γ, E, T, D, t) of the particles with rotational diffusion coefficient D at moment t and by an optical function G(L/λ, n1/n0), which depends on the form, the relative size L/λ at wavelength λ in the medium, and the relative refractive index n1/n0 of the cells n1 and the medium n0. In steady-state EOE (∆As) the F(γ, E, T) depends only on the ratio between the ∆A/A0) is orientation energy γE2 and the energy of random motion kT and then the relative value of the EOE (∆A/A0) is

∆As /A0 ) G(L/λ, n1 /n0)(γE2 /15kT)

(1)

Equation 1 is valid at low degrees of orientationswhen γE , kT. The EOE decay after the switching off the electric field is defined by D, the relaxation time τ ) 1/6D. The EOE decay of a monodisperse suspension of particles with axial symmetry is a monoexponential function of time t: 2

∆At ) ∆As exp(-6Dt) ) ∆As exp(-t/τ)

(2)

where ∆At and ∆As are the values of the EOE at the moment t and at the steady state, respectively. The frequency dependence of the EOE was measured by the EloTrace 1.0, developed in Biotronix GmbH (Germany). The device records the EOE as ∆A ) ∆A| - ∆A⊥, where ∆A| and ∆A⊥ are the values of the EOE at parallel and perpendicular orientation of two light beams with respect to the electric field. Because ∆A| and ∆A⊥ have an opposite sign (as a result of different inner destructive interference because of the orientation of bacterial cells parallel or perpendicular with respect to the light wave), the resulting EOE is a sum of both the components. The device takes down automatically the dependencies of the EOE on the frequency (20 kHz to 20 MHz) and the electric field intensity (17-110 V/cm) at wavelength 670 nm and optical path 1 cm. It also calculates γ and τ according to eqs 1 and 2 and the average size of the bacteria. The size and the polarizability of the cells during the growth were recorded by the EloTrace 2.0 (Biotronix GmbH, Germany), which has the same electrical and optical characteristics but makes all the operations completely automatic. The device takes a bacteria sample of 1-2 mL from the incubator every g6 min, filters, washes, suspends, and dilutes it until obtaining a suspension with an optical density 0.1 and electric conductivity 5 µS/cm. After that the EloTrace 2.0 takes down and treats automatically the electro-optical data so that the electric polarizability, the average size, the growth time, and other parameters can be monitored and recorded. All the frequency dependences in this work were taken down at an electric field strength of E ) 78 V/cm, at which eq 1 is valid, the evidence for which is the linear dependence of ∆As on E2.

Maxwell-Wagner Polarizability of Bacteria

Figure 1. Frequency dependencies of electric polarizability γ (in relative units) of E. coli K12 (fixed by formaldehyde) in aqueous ethanol medium: 5 mL/dL ethanol (2), 10 mL/dL ethanol (3), 15 mL/dL ethanol (4), 20 mL/dL ethanol (5), and control sample without ethanol (1).

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Figure 2. Dependence of the electric polarizability γmax (in relative units at the maximum of frequency dependences) of E. coli K12 on the ethanol concentration for alive (1) and fixed by formaldehyde (2) bacteria.

3. Results and Discussion Figure 1 shows the dependence of the electric polarizability γ of E. coli K12 fixed by formaldehyde on the frequency of the electric field applied in an aqueous suspension and in aqueous ethanol suspensions. The value of γ is automatically calculated by the used device from the steady-state value of the EOE using eq 1. As seen, the polarizability decreases in presence of ethanol at all the frequencies, where a highfrequency polarization is shown up (a bacteria orientation is not observed above 2 MHz). The decrease in the maximum of the dispersion curve is from 20% to 50% at ethanol concentration from 5 to 20 mL/dL, respectively. The decrease of γ in Figure 1 could be due to a change in the refractive index, the dielectric permittivity, and the ionic strength of the medium. 3.1. Refractive Index and Dielectric Permittivity. The refractive index of the medium n0 increases from 1.331 to 1.345 (at 670 nm, 20 °C) with the increase in the ethanol concentration from 0 to 20 mL/dL,18 which leads to decrease in the light scattering intensity19 and correspondingly in the optical density A of the suspension. However, the calculations based on the theories of Mie20 and Rayleigh-Debye-Gans21 show that the increase in (n0 - 1) by 4% causes an insignificant change in A (at an invariable geometry of the particles). The change in n0 influences negligibly the value of γ, which is calculated from the relative value of the EOE ∆As/A0 ) AE/A0 - 1, because the optical densities AE and A0 are influenced in an almost equal degree by n0 in the cases of bacteria orientation in electric field and at random orientation, respectively. This insignificant difference is related to the change in the optical function G(L/λ, n1/n0) (eq 1) because of the alteration in both the relative refractive index n1/n0 and the relative size L/λ, where the wavelength in the medium is λ ) λ0/n0. However, this effect is too weak and could not explain quantitatively the decrease in the polarizability by one-half in Figure 1. That conclusion is valid only if the geometrical dimensions of the cells are not significantly influenced by the presence of ethanol. An experimental proof for the constancy of the size is presented below. Information for the character of the ethanol effect could be given by the dependence of the electric polarizability on the ethanol concentration γ(C). Figure 2 shows that this dependence is linear. That leads to the supposition that the decrease in γ may be related to a change in the relative dielectric permittivity

Figure 3. Dependence of the relative dielectric permittivity (line εrel, left ordinate) of the water-ethanol solution (follow ref 22) on ethanol concentration Cethanol at 25 °C. Dependences of EDL thickness (curves 1 and 2, right ordinate) on ethanol concentration Cethanol (curve 1, bottom abscissa, at ionic strength 0.1 mM) and on KCl concentration CKCl in water medium (curve 2, top abscissa, at εrel ) 78.25).

εr, which concentration dependence εr(C) is linear too (Figure 3). The dielectric permittivity decreases from 78 to 67 (at 25 °C) with the increase in the ethanol concentration from 0 to 20 mL/dL.22 The change in εr influences both the ChD and the MW components of polarizability. The decrease in εr leads to decrease in the thickness of the EDL and consequently to decrease in the ChD polarizability. The thickness of the EDL diminishes by 7% (at 0.1 mM KCl) according to the Poisson-Boltzmann equation,23,24 when the ethanol concentration increases from 0 to 20 mL/dL (Figure 3, curve 1). This decrease is equivalent to the decrease in the EDL thickness with the increase in the electrolyte concentration (in absence of ethanol) from 0.1 to 0.115 mM KCl (Figure 3, curve 2). Such an increase in the KCl concentration causes a decrease in γ (at the maximum of the frequency dependence) by 15% according to our previous results.15 That decrease is too small to explain the decrease in γ by one-half in presence of ethanol (Figures 1 and 2). The decrease in εr leads to decrease in the value of the MW polarizability, because the difference between the permittivity of the aqueous ethanol medium and the lipid membrane

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decreases. The calculations of the EPh model for a homogeneous ellipsoid show that the polarizability decreases by one-third with the decrease in εr of the outer medium from 80 to 60 (in the frequency range from 103 to 107 Hz).3 Despite such a decrease is lower than that in our experiment, the evaluation shows that the change in εr may have a significant contribution to the decrease in γ in presence of ethanol. 3.2. Kinetics of the Polarizability and the Size. The possible influence of the permittivity could be checked by investigation of the kinetics of the change in γ, because the effect of the ethanol on εr and n0 is observed immediately after adding ethanol. However, the result in Figure 4 shows that the changes in γ are too slow and continue some hours. This fact excludes the possibility that the decrease in γ may be due to the direct influence of the ethanol on the electric (εr) and the optical (n0) properties of the outer medium. The slow change in the value of γ indicates that the reason may be related to diffusion of ions and ethanol through the lipid membrane. The commensurable kinetics of the decrease in γ in presence of ethanol and in its absence (Figure 4) shows that the effect of the ethanol is not due to a decrease in εr of the cytoplasm as a result of an infiltration of the ethanol in the cells. So, the change in the ionic concentration because of the passive transport of ions through the lipid membrane remains the most probable cause for the change in γ. The difference in the electrolyte concentration in the inner and the outer medium (Figure 4) is created by incubation of the bacteria in 0.15 M KCl for 24 h and replacement of this medium by distilled water just before the electro-optical experiment. The diffusion through the cell membrane is accelerated in presence of ethanol because of increased permeability of the lipid bilayer.16,17 This supposition is confirmed by the kinetics in both the cases presented in Figure 4. The higher initial value of γ after the incubation in presence of ethanol shows that the cytoplasm electrolyte concentration is increased stronger in this case. This ratio is changed after the second hour of the incubation in distilled water because of the faster diffusion through the ethanol damaged membrane. The bacteria swell up after their removal from a medium with 0.15 M KCl to distilled water because of the increased osmotic pressure. This swelling results in increased linear dimensions of the cells, but no more than 10-15% according to the literature.3 Figure 5 confirms these data in the opposite caseswhen the osmotic pressure decreases, the decrease in the average bacteria size does not exceed 10%. The reasons for the limited osmotic swelling of the bacteria are the high module of elasticity of the cell wall and cytoskeleton,25,26 as well as the presence of mechanosensitive channels27,28 in the cytoplasm membrane. These channels are open at reaching a definite membrane tension, so that the osmotic pressure is maintained within definite borders because of the flowing of some quantity of ions to the outer medium. This conception is corroborated by the two-speed dependence of the average bacteria dimension on the time (Figure 5). It could be supposed that this dependence expresses both the mechanisms of ionic transport through the membranesby the mechanosensitive channels (the faster component) and the passive diffusion through the lipid bilayer (the slower component). 3.3. Influence of the Size on the Polarizability. The decrease in the concentration gradient with the time leads to decrease in the osmotic pressure followed by decrease in the bacteria size. This results in decrease in the induced dipole moment and the degree of orientation because of the decrease

Zhivkov and Gyurova

Figure 4. Time dependence of electric polarizability (in relative units) of E. coli K12 (fixed by formaldehyde) at 160 kHz in distilled water after 24 h of incubation in 150 mM KCl with added 5 mL/dL ethanol (curve 1) or without ethanol (curve 2).

Figure 5. Time dependence of the average size of E. coli K12 (fixed by formaldehyde) in distilled water after 24 h of incubation in 150 mM KCl with added 5 mL/dL ethanol (curve 1) or without ethanol (curve 2).

in the electric polarizability. This effect is especially expressive on the ChD polarizability, which is proportional to the second power of the length of rodlike particles.5,7 This is a possible reason for the decrease in γ in presence of ethanol (Figures 1 and 2). The electro-optical method suggests a reliable check of this possibility, because the coefficient of the rotational diffusion D depends on the third power of the longest bacteria dimension according to the Perrin’s equation.29,30 Therefore, the time of disorientation τ (eq 2) is the most sensitive parameter regarding the size of the particles. Furthermore, τ (after the switching off the electric field) does not depend directly on the electric properties of the particles. Figure 5 shows the time dependence of the average size (calculated by the used apparatus) of the bacteria suspended in distilled water after their previous incubation in 150 mM KCl. The recorded decrease in the average size by 9% could lead to decrease in the ChD polarizability by about 18% in the case when only the bacteria length is changed. However, it may be supposed that the change in the cell volume is accompanied by a corresponding change in both the dimensions. Because the polarizability is determined by the difference between its values along the longer (γ|) and the shorter (γ⊥) axes of the particle, γ ) γ| - γ⊥, the decrease in the diameter compensates in some degree the effect of the decrease in the length. Furthermore, the decrease in γ would be even weaker if the increase in the

Maxwell-Wagner Polarizability of Bacteria

Figure 6. Time dependences of the average size (curve 1) and the electric polarizability at 160 kHz (curve 2) of E. coli K12 (fixed by formaldehyde) in distilled water after 24 h of incubation in 150 mM KCl with added 5 mL/dL ethanol. Both curves are set to unity at t ) 0.

surface charge density (as a result from the osmotic compression of the previously swelled bacteria) is taken into account. On the other hand τ decreases with the decrease in both the dimensions, because D is sensitive to the length (at the third power), as well as to the diameter (at small axial ratio, which is about 3 for E. coli K12). Therefore, the change in the average bacteria size (at constant axial ratio) must change more strongly τ and more weakly γ. This conclusion is also valid when only the bacteria length is changed because of the stronger dependence of τ on the longer axis L of the particles (τ ∼ L3) compared to that of γ on L (γ|ChD ∼ L2), i.e., ∆τ/∆γ|ChD ∼ ∆L in the case of the longitudinal component γ|ChD of the charge dependent polarizability. For MW one the ratio ∆τ/∆γ| is even more significant because its value γMW is proportional to the volume of the particle [1], so γ|MW ∼ L and ∆τ/∆γ|MW ∼ (∆L)2 at constant diameter if approximate E. coli to cylinder. If only the bacteria diameter decreases, even the opposite dependence should be observedsthe decrease in τ would be accompanied by increase in γ ) const – γ⊥ in both the cases of ChD and MW polarizabilities because the weak dependence of τ on the diameter d and the quadratic ones of both components γ⊥ChD ∼ d2 and γ⊥MW ∼ d2. The kinetics in Figure 6 shows the opposite of the three cases described above (ratio ∆γ/∆τ when the bacteria change only their length or diameter, or both the dimensions)s3-fold decrease in γ (for 260 min) at insignificant decrease in the average cell size. This allows us to make the conclusion that the decrease in γ, observed in Figures 2 and 4, is not due to the change in the geometrical dimensions of the bacteria. Consequently, the cause for the change in 2 must be searched in the electrical aspects of the change in the electrolyte concentration but not in the osmotic pressure. 3.4. Extracellular Electrolyte Concentration. The decrease in the inner electrolyte concentration and its increase in the outer medium should result in decrease in the iMW and eChD the components and increase in the iChD and the eMW components of the polarizability (independently from the mechanism of the ionic flow through the two lipid membranes). The 3-fold decrease in the total bacteria polarizability γ (Figure 6) shows that the iMW component determines in a high degree the cells’ orientation in electric field at 160 kHz. The determination of the ionic concentration in the inner and the outer medium is necessary to evaluate the contribution of the rest components. The change in the electrolyte concentration caused by the flowing of ions out of the bacteria could be estimated by the

J. Phys. Chem. B, Vol. 113, No. 24, 2009 8379 ratio between the total volume of the cells and that of the outer medium. The bacteria concentration is 107 cells/mL (at optical density 0.1 in our experiment), and the volume of a cell of length 3 µm and diameter 1 µm is equal to 2.1 µm3. If all the ions from the bacteria (with total volume of 2 × 107 µm3) are distributed homogeneously in 1 cm3, their concentration would decrease (5 × 104)-fold compared to the initial value. It could be accepted that the initial cytoplasm electrolyte concentration is of the order of 0.15 M because the cells have grown in a medium with such a concentration of NaCl. If all the ions flow out, their concentration in the outer medium would increase only by 3 µM. This is an order less than the initial concentration when the bacteria are suspended in distilled water (the conductivity 5 × 10-4 S/m corresponds to 3 × 10-5 M KCl or 4 × 10-5 M NaCl). We found out in our previous work15 that the ionic strength influences appreciably the value of γ at concentration above 0.1 mM KCl. Therefore 30-fold lower alteration in electrolyte concentration cannot change the eChD polarizability. The EPh model for homogeneous ellipsoid shows that the eMW component is shown up at electric conductivity above 10-2 S/m,3 which corresponds to 7 × 10-4 M KCl or 8 × 10-4 M NaCl. That means that the eMW polarizability is not shown in our case, because the bacteria are suspended in electrolyte solution with 20-fold less concentration. Consequently, both the outer components of polarizability (eChD and eMW) are not related to the observed decrease in γ of the cells in aqueous medium after adding ethanol (Figures 1 and 2) and after incubation in 0.15 M KCl (Figure 4). 3.5. Cytoplasmic Electrolyte Concentration. Figure 4 shows that the electric conductivity of the cytoplasm decreases with the time after the bacteria were suspended in distilled water. If the inner electrolyte concentration gets lower than 1 mM, the iMW component would disappear and the iChD one would be shown up. The ionic concentration inside the cells could be evaluated, if we accept that the iMW polarizability is proportional to the electrolyte concentration. This approach is applicable, if the change in the total polarizability is determined only by the change in the iMW component, as shown in the present work. The dependence of γ on the ethanol concentration is linear for alive bacteria, as well as for the fixed ones (Figure 2). This indicates that the fixation with formaldehyde does not change the ability of the ethanol to increase the permeability of the lipid membrane. Therefore, the speed of flowing of the ions should be proportional to the concentration gradient. The smaller slope of the time dependence of γ for the fixed bacteria (Figure 4) shows that the electrolyte concentration in the fixed cells is lower than that in the alive cells, possibly because of the longer stay of the fixed bacteria in the water before the electro-optical experiment. However, the difference is not large, which allows us to conclude that the ionic concentration in the dead bacteria is of the same order as that in the alive cells. A possible reason for this is that some of the ions in the inner medium cannot leave it because of the electrostatic interactions with the macromolecule components of the cytoplasm and the membrane. The close quantity of γ of the alive and the fixed bacteria is not enough to find out if the cytoplasm electrolyte concentration is high or low by absolute value after the stay in the distilled water. Such a conclusion could be made taking into account the values of γ and its kinetics. The slow decrease in γ (Figure 4) indicates that the inner electrolyte concentration is nearly not changed in the beginning of the electro-optical measurement

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(about 10 min after the washing of the bacteria with distilled water). The values of γ of fixed bacteria in aqueous medium in absence of ethanol nearly coincide after a long stay in distilled water (Figure 2, line 2 at C ) 0) and after 24 h of incubation in 150 mM KCl (Figure 4, curve 2 at t ) 0). Therefore, the inner electrolyte concentration is practically equal in both the cases. If we accept that the speed of the ionic transport through the membrane is commensurable in both the directions (into and out of the cells), the kinetics of polarizability (Figure 4, curve 2) indicates that 24 h is enough time to achieve equal electrolyte concentration in the inner and the outer medium (0.15 M KCl). So, we can assume that the electrolyte concentration in the cytoplasm is about 0.1 M in the beginning of the electrooptical measurements (NaCl in the case of Figures 1 and 2 or KCl in Figures 4-7). We found out that the electric polarizability decreases 6-fold with the increase in the ionic concentration of the outer medium from 0 to 1 mM KCl (at constant cytoplasm electrolyte concentration).15 The ChD polarizability practically is not shown at 2 orders higher ionic concentration. This means that the iChD component has not any contribution to the total polarizability in both the cases of alive and fixed bacteria (where the inner electrolyte concentration is approximately 0.1 M). The EPh model for a homogeneous ellipsoid shows that the concentration of univalent electrolyte inside the cell influences γ at an electrolyte conductivity of 0.06-0.6 S/m (in the frequency range of 104-108 Hz).3 This conductivity value corresponds to 4-46 mM KCl or 5-54 mM NaCl (at 25 °C). The comparison to the inner ionic concentration of 0.1 M, evaluated above, presents that the electric conductivity inside the fixed bacteria is high enough for manifestation of the iMW polarizability. The above estimation of the contribution of the four polarizability components allows us to conclude that the decrease in γ (Figures 1, 2, 4, 6) is due to the change in the iMW component resulting from the decrease in the cytoplasm electrolyte concentration. The increase in the inner ionic strength of the fixed bacteria is a check of this conclusion, because the increase in γ with the increase in the inner conductivity results in increasing the MW polarizability in contrast to the decreasing the ChD one. Figure 7 presents that the incubation of the cells in 150 mM KCl increases the inner conductivity, which is proved by the increase in γ after adding the ethanol. In this case the effect of the ethanol is opposite to that observed in Figure 1, when the ethanol is added to an aqueous medium and the cytoplasm electrolyte concentration decreases. The opposite influence of the ethanol on γ in these two cases is a proof that its effect is related to the iMW component because of a change in the inner electrolyte concentration. 3.6. Relaxation Frequency. Another confirmation of the role of iMW polarizability is the change in γ at different frequencies. Because the relaxation frequency of the MW polarizability is in several orders higher than that of the ChD polarizability,3 the change in the iChD component should appear predominantly at the lower frequencies, whereas the change in the iMW component must be observed in the whole range up to megahertz frequencies. As shown in our previous work,15 the increase in the outer ionic strength leads to stronger decrease in γ at the lower frequencies, which proves a ChD mechanism of polarization on the outer bacteria surface. On the contrary, Figure 7 shows that the value of γ is higher (at higher inner ionic strength) in the whole frequency range, which is a proof for a MW polarization on the inner membrane surface.

Zhivkov and Gyurova

Figure 7. Frequency dependence of electric polarizability γ (in relative units) of E. coli K12 (fixed by formaldehyde) in distilled water after 24 h of incubation in 150 mM KCl with added 5 mL/dL ethanol (curve 2) or without ethanol (curve 1).

Figure 8. Dependence of frequency of the maximum (line νmax, left ordinate) and relaxation frequency (line νmax/2, right ordinate) on ethanol concentration Cethanol of E. coli K12 (fixed by formaldehyde). The data are obtained from dispersion curves on Figure 1.

The increase in the ionic strength shifts the relaxation frequency to higher values in both the cases of the ChD polarizability31 and the MW one.4 As mentioned above, the eMW and the iChD components are not shown up and the eChD component does not change under the conditions of our experiment (low outer and high inner electrolyte concentration). Therefore, the changes in the frequency characteristics of γ show only the contribution of the iMW component. So, the shift of the maxima of the curves in Figures 1 and 8, and the shift of the relaxation frequency νmax/2 (determined as the frequency at which γ ) γmax/2, Figure 8) to lower frequencies, reflects the decrease in the inner electrolyte concentration. Figure 7 presents the oppositesshift of the maximum to higher frequencies at higher inner electrolyte concentration. Figure 7 is in an agreement with the conclusion that the ethanol effect is not related to changes in the dielectric permittivity, which would decrease both the eChD and the iMW components. The same conclusion could be made also for possible conformational changes in the structural components of the bacteria caused by ethanol. The kinetics in Figure 4 shows that the inner electrolyte concentration decreases in absence of ethanol too, but its presence accelerates this process. Consequently, the ethanol effect could be explained with an increase in the permeability of the two lipid membranes of E. coli, which results in faster passive ionic transport. This leads to decrease

Maxwell-Wagner Polarizability of Bacteria or increase in the inner electrolyte concentration depending on the direction of the concentration gradient and correspondingly to decrease or increase in the iMW component of polarizability. 3.7. Possible Electrodeformation of the Bacteria. The accumulation of free ions on both the sides of the bacteria membrane (MW polarizability) leads to deformation forces (Maxwell stresses).32–35 This may lead to deformation of the cells. The degree of deformation depends on their mechanical properties and the intensity of the applied electric field. Such deformation is electro-optically detected for erythrocytes36 and purple membranes.37 The purple membranes (fragments from the bacterial cytoplasm membrane) differ from the cells in their shape of plates, which do not close any volume. That is why their deformation is determined only by the deformation force and the modulus of elasticity at bending. In weak electric field the purple membranes orientation is like that of rigid plates, whereas at intensity above 2.5 kV/cm an orientation-deformational effect is observed.38 The deformation leads to two-phase kinetics of the decay of the EOE after the switching off the field so that the faster relaxation effect is related to the membranes’ shape recovery and the slower one to their disorientation. In the case of biological cells the deformation depends on that if the change of the cell form requires some change in the cell volume. In the case of erythrocytes the form can be changed so that the volume remains constant; therefore, they are easily deformable. Nevertheless, deformation of red blood cells is practically missing up to 0.4 kV/cm according to ref 39. The value of the external electric field used in our experiments with E. coli K12 is less than 0.1 kV/cm. Bacteria deformation requires some change in the cell volume, so it depends on the membrane permeability. In our previous work15 it was shown that in the case of E. coli two-phase kinetics of the EO decay is not recorded; consequently, the bacteria orientation is similar to that of solid particles. The main two reasons for that are the low electric field intensity and the slow passive transport through the membrane. The increased membrane permeability under the action of ethanol would make the deformation possible; however, we observe neither the mentioned two-phase kinetics of the EO decay nor any change in the time of disorientation after series of electric pulses at E ) 78 V/cm and ethanol concentration up to 20 mL/dL. So, our conception is that electrodeformation is missing under the conditions of the experiment and EOE is determined only by electro-orientation of the bacterial cells. 4. Summary The value of the electric polarizability γ decreases in the whole frequency range from 20 kHz to 2 MHz in presence of ethanol in aqueous suspension of E. coli K12. This decrease in γ reaches 50% at 20 mL/dL ethanol at the maximum of the dispersion curve and depends linearly on the ethanol concentration for alive bacteria, as well as for bacteria fixed by formaldehyde. The decrease in γ is accompanied by a shift of the maxima of the dispersion curves (from 200 to 80 kHz) and the relaxation frequency of γ (from 660 to 290 kHz). The polarizability decreases by a factor of 2 for 4 h in distilled water after preliminary incubation in 0.15 M KCl. The initial value of γ in distilled water is higher when 5 mL/dL ethanol is present in the preincubation medium with 0.15 M KCl, but γ decreases faster (2-fold for 2 h) in comparison with the case of absence of ethanol in the KCl medium. The quantitative estimation shows that the ethanol effect is not related to changes in the refractive index and the dielectric

J. Phys. Chem. B, Vol. 113, No. 24, 2009 8381 permittivity of the outer and the inner medium. The slow kinetics of changing γ and the opposite effect of the ethanol (decreasing and increasing γ) in aqueous suspension and in 0.15 M KCl, respectively, confirm the literature data that the ethanol increases the ionic permeability of the cytoplasm membrane. The intracellular electrolyte concentration of the bacteria in distilled water decreases but remains high enough to keep the contribution of the iChD polarizability negligible. The increase in the extracellular ionic concentration caused by the flowing of the ions out of the cells is insignificant and cannot influence the eChD component. The eMW polarizability is not shown up at such a low extracellular electric conductivity. The osmotic pressure cannot change both the ChD and MW components of the polarizability because the change in the linear dimensions of the bacteria does not exceed 10%. So, the change in the iMW component because an intracellular conductivity alteration remains the only real reason for the observed changes in γ. It is found that only the inner MW and the outer ChD components of the polarizability of E. coli K12 are shown up in the frequency range from 20 kHz to 2 MHz. The conclusion is made that the decrease in the total polarizability in aqueous medium (in presence and absence of ethanol) is due to the contribution of the iMW component because of the decrease in the cytoplasm electrolyte concentration. This is confirmed by the decrease in the relaxation frequency, which accompanies the decrease in the value of the polarizability. The influence of the ethanol is explained by the increase in the ionic permeability of the two lipid membranes and the caused by that acceleration of the passive ion transport through them. Acknowledgment. We thank Dr. A. Angersbach and Dr. V. Bunin for the possibility, which they gave us, to make the experiments in the laboratory of Biotronix GmbH, Germany, using the automatic electro-optical devices EloTrace 1.0 and EloTrace 2.0 developed in the same company. References and Notes (1) O’Konski, C. T. Theory of Kerr constant. In Molecular ElectroOptics; Krause, S. Ed.; Plenum Press: New York, 1981. (2) Schwan, H. P. AdV. Biol. Med. Phys. 1957, 5, 147. (3) Miroshnikov, A. I.; Fomchenkov, V. M.; Ivanov, A. Yu. ElectroPhysical Analysis and Separation of Cells; Nauka: Moscow, 1986. (4) Dukhin, S. S.; Shilov, V. N. Dielectric Phenomena and Double Layer in Disperse Systems and Polyelectrolytes; Wiley: New York, 1974. (5) Stoylov, S. P.; Shilov, V. N.; Dukhin, S. S.; Sokerov, S.; Petkanchin, I. Electro-Optics of Colloids; Dukhin, S. S. Ed.; Naukova Dumka: Kiev, 1977. (6) Buleva, M.; Stoimenova, M. J. Colloid Interface Sci. 1991, 141, 426. (7) Stoylov, S. P. Colloid Electro-Optics: Theory, Techniques and Applications; Academic Press: London, 1991. (8) Jones, J. F.; Feick, J. D.; Imoudo, D.; Chukwumah, N.; Vigeant, M.; Velegol, D. Appl. EnViron. Microbiol. 2003, 69, 6515. (9) Asami, K.; Hanai, T.; Koizumi, N. Biophys. J. 1980, 31, 215. (10) Takashima, S. Electrical Properties of Biopolymers and Membranes; Adam Hilger: Bristol, U.K. and Philadelphia, PA, 1989. (11) Peikov, V.; Stoylov, S.; Petkanchin, I. J. Colloid Interface Sci. 1995, 1172, 389. (12) Dimitrov, V.; Stoimenova, M.; Tsoneva, J. Colloids Surf., A 2002, 209, 201. (13) Volk, W. A. Basic Microbiology; Harper Collins: New York, 1992. (14) Hanai, T.; Koizumi, N.; Irimajiri, A. Biophys. Struct. Mech. 1975, 1, 285. (15) Zhivkov, A. M.; Gyurova, A. Y. Colloids Surf., B 2008, 66, 201. (16) Angersbach, A.; Bunin, V.; Ignatov, O. Electro-optical analysis of bacterial cells. In Molecular and Colloid Electro-Optics; Stoylov, S. P., Stoimenova, M. Eds.; Taylor & Francis: New York, 2006. (17) Gurova, A. Y.; Zhivkov, A. M. Biophys. Chem. 2009, 139, 8. (18) Ioffe, B. V. Refractometric Methods in Chemistry; Himia: Leningrad, 1983. (19) van de Hulst, H. C. Light Scattering by Small Particles; John Wiley: New York, 1957.

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Zhivkov and Gyurova (31) Petkanchin, I. B. Counterions dynamics as studied by electric light scattering. In Molecular and Colloid Electro-Optics; Stoylov, S. P., Stoimenova, M. Eds.; Taylor & Francis: New York, 2006. (32) Bryant, G.; Wolfe, J. J. Membr. Biol. 1987, 96, 129. (33) Helfrich, W. Z. Naturforsch. 1974, 29, 182. (34) Pawlowski, P.; Fikus, M. J. Theor. Biol. 1989, 137, 321. (35) Winterhalter, M.; Helfrich, W. J. Colloid Interface Sci. 1988, 122, 583. (36) Dzenev, I.; Petrova, R.; Stoylov, S. Cell Biophys. 1990, 16, 161. (37) Zhivkov, A. M. Geometry of purple membranes in aqueous medium. In Molecular and Colloidal Electro-Optics; Stoylov, S. P., Stoimenova, M. V. Eds.; Taylor & Francis: New York, 2006. (38) Zhivkov, A. M. Colloids Surf., A 2002, 209, 327. (39) Sukhorukov, V. L.; Mussauer, H.; Zimmermann, U. J. Membr. Biol. 1998, 163, 235.

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