19620 IRE TRANSACTIONS ON BIO-MEDICAL ELECTRONICS 93

Electrical Properties of the Cytoplasmic Membrane

and the Cytoplasm of Bacteria
and of Protoplasts*
HELMUT PAULYt, M.D.

INTRODUCTION in the case of a cell by the relatively high conductive proto-

r HE OBJECT of this investigation is the bacterial plasma and the external medium. These are usually much
cell of micrococcus lysodeikticus and the proto- more conductive than the lipid layer of the membrane.
plasts obtained by lysozyme treatment of the micro- Since it is not possible to measure the membrane capac-
coccus. ity in a direct way by introducing one electrode into the
The micrococcus is a spherical cell with a diameter of bacteria cell or the protoplast, with another one outside,
0.75 ,u. The cell wall is 550 A thick. This wall can be an indirect way of measurement had to be chosen. This
dissolved by enzymatic digestion with lysozyme. What re- was done by measuring the frequency dependence of the
mains is a cytoplasmic spherical body called the protoplast. dielectric constant e and conductivity x of a suspension of
This protoplast is surrounded by an osmotic barrier [8] bacteria and protoplasts. The dielectric constant and con-
which is shown to be the cytoplasmic membrane. ductivity were measured in the frequency range from 0.5-
The problems are: 250 Mc with a Schering bridge.
1) to measure the electrical capacity of the membrane
of the micrococcus,
2) to compare this value with the capacity of the proto-
plast membrane, and
3) to measure the electrical conductivity of the cyto-
plasm of the cell and of the protoplast and relate the
value to the well-known very high osmotic pressure Fig. 1-Membrane condenser.
within the cell.
A number of contributions, predominantly due to Fricke LOW FREOUENCY HIGH FREQUENCY
[6], Cole [5], Cole and Curtis [2], [3], and Schwan [14],
[15], [16], show that the electrical capacity of all investi- -11z-,
gated cell membranes is near 1Jf/cm2 [14]. This applies
to cells such as nerve fibres [3], erythrocytes [6], marine
eggs [1], bacteria [7], plant cells [2] and mitochondria
---------------
[12], [13]. This report shows that the protoplast mem-
brane of the micrococcus has nearly the same electrical
capacity as the cell membranes previously investigated.
The electrical capacity of a condenser with an area of
1 cm2 is (Fig. 1) 4

CM= =
- (Farad/cm2).
4- 4, 4
d Fig. 2-Frequency dependence of path of current flow around and
Cm is proportional to the ratio of the dielectric constant X through a spherical cell and of induced dipole moment.
of the material between the electrodes divided by the dis-
tance d; e is the dielectric constant of the vacuum and a
, The behavior of a cell in an alternating field is shown
known number. The corresponding membrane condenser in Fig. 2. At low frequency the electric current is forced
is shown on the left side of Fig. 1. It is a schematic draw- to flow around the cell on account of the high resistance
ing of the cellular membrane. The lipid layer acts as a of the membrane. With increasing frequency the mem-
dielectric of high electrical resistance. The task of the brane capacitance becomes more and more short-circuited,
electrodes and the current conducting cables is performed until at high frequency the interior contributes fully to
the conductivity. Hence, the electrical conductance must
* Received by the PGBME, November 3, 1961.
t Max-Planck-Institut fur Biophysik, Frankfurt am Main, Ger- increase with increasing frequencies. The opposite be-
many. havior is exhibited by the dielectric constant as can be

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94 94 IRE TRANSACTIONS ON BIO-MEDICAL ELECTRONICS A pril

seen in the lower row of the figure. In the case of low 714"- 3
frequencies, dipoles are built up by the applied electrical 400 I,
- -. .a 1

11K
field. This is due to the fact that the field separates the
positive and negative ions which accumulate near the
highly resistive membrane. Since the movement of the ions
needs time, the separation of the ions becomes less and
I 300F

200F
10-1-
I9V

less complete with increasing frequency of the alternating

field. Hence the dielectric constant will decrease as the fre- 100l 7I' I
quency increases. 6
f, =
11 III
RESULTS l 10 100 MHz
Frequenz
One of the measured dispersion curves of a suspension Fig. 3-Frequency dependence of the dielectric constant e and the
of the intact cell is shown in Fig. 3. The frequency is specific conductivity x of intact cells suspended in a medium con-
taining 0.1 M KCI and 0.5 M saccharose. Volume fraction 0.31.
plotted on the abscissa from 0.5-200 Mc. As expected the
dielectric constant decreases as the frequency increases.
The conductivity shows the opposite behavior. The analy-
sis of these curves was done by means of appropriate 150
150
-

potential theory, using the Cole-Cole-plot [4I, shown in foE 23 MHz

Figs. 4 and 5. Fig. 4 shows the plot of the imaginary part $100
of the DK vs the real part of DK in order to obtain the CJEr %
value for the dielectric constant at zero frequency. Fig. 5 5
so
shows the plot of the imaginary part of the conductivity
vs the real part of the conductivity X in the admittance
plane, in order to obtain the value of X of infinite high 0o 1 50 to00 M 200 250 1300
frequency. &,-34 6 fo -261
The analysis [14], [9] gives the electrical capacity of
the membrane of intact cells of nearly 1 Vf/cm2. This is Fig. 4-Plot of x - xp/w, vs the dielectric constant E in the
complex dielectric plane. x. is the LF conductivity of the sus-
the same value found for all other cells and intracellular pension, X = 2rrQ, and Q the frequency in cps. Frequencies are
particles. Especially for E. coli cells, this has been shown indicated in megacycles. The half-value frequency of the repre-
sentation in the complex dielectric plane Qo. is 28 Mc (Mc=
by Fricke, Schwan, Li, and Bryson [7]. MHz).
After removing the cell wall, the dielectric behavior of
the protoplast is shown in the next two figures. Fig. 6 is
similar to the corresponding dispersion curve of intact
cells. Fig. 7 shows the corresponding behavior in the ad- 3 [s2rcmi]
mittance and the dielectric plane. ,I ; _ 45MHZ
The analysis gives the same electrical capacity of the
2 0
protoplast membrane of nearly 1 Vtf/cm2. Hence we con-
-

clude that the membrane of the protoplast is the well-

known cytoplasmic membrane. It is the same membrane
which determines the dielectric behavior of the intact mic-
rococcus. After digesting the cell wall, the thin bimolecular 05 7 8 9 10 1 12 lo0-3
leaflet of the cytoplasmic membrane did not become altered K0o59 \ K [N`cm-7J w
in a measurable way. K.t11,7
The third problem was concerned with the electrical con- Fig. 5-Plot of (0)e, (e - ee) vs x in the complex admittance plane.
ductivity of the cytoplasm itself. The electrical conductiv- e, is the HF dielectric constant. Frequencies are indicated in
megacycles. The half-value frequency of the representation in the
ity of an electrolyte is determined by the number of ions complex admittance plane Q., is 45 Mc (Mc = MHz).
per volume unit and by their mobility. Therefore, the
electrical conductivity of the cell interior or the cell proto- in most of the cells of mammals; it is three times higher
plasm should provide information about the number of than in red blood corpuscles as shown by Schwan and his
ions within the cell, and whether they are bound or free- co-worker [10], before. The three times higher conductiv-
moving. ity in the bacteria is mainly due to a nearly three times
The analysis of the measured dispersion curves shown higher concentration of ions, which was shown by dissolv-
before yields a value of nearly 15 mmho/cm for thie in- ing the cells in a large volume of distilled water and ap-
ternal conductivity in intact cells. After lysozyme treat- proaching the state of an "ideal electrolyte" [10]. The
ment in the proper medium, nearly the same value was higher concentration is in agreement with the fact that
obtained with the protoplasts. This value is higher than the osmotic pressure of this kind of cells is around 20

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1962 Pakly: Electrical Properties of Cyptoplasmic Membrane 95

CONCLUSIONS
1) The micrococcus cell has the cytoplasmic membrane
of 1 pIf/cm2.
2) This membrane is not altered measurably by remov-
ing the cell wall.
3) The ion concentration within the cell is very high.
4) The average mobility of the ions is reduced by a
factor of 2.
BIBLIOGRAPHY
[1] K. S. Cole, "Electric impedance of marine egg membranes,"
Fig. 6-Frequency dependence of e and x of protoplasts, suspended Nature, vol. p. 141, p. 79; 1938.
in a medium containing 1 M. saccharose and 0.04 M phosphate- [2] K. S. Cole and H. J. Curtis, "Electric impedance of nitella
buffer pH 6.8. during activity," J. Gen. Physiol., vol. 22, pp. 37-64; 1938.
[3] - , "Electric impedance of the squid giant axon during ac-
tivity," J. Gen. Physiol., vol. 22, pp. 649-670; 1939.
[4] K. S. Cole and R. H. Cole, "Dispersion and absorption in di-
o11 .CAE electrics. I. Alternating current characteristics," J. Chem.
Phys., vol. 9, pp. 341-351; 1941.
[5] K. S. Cole, "Impedance of single cells," Tabulae Biologicae,
vol. 19, pars. 2, 1942.
[6] H. Fricke, "The electric capacity of suspensions with special
reference to blood," J. Gen. Physiol., vol. 9, pp. 137-152; 1926.
[7] H. Fricke, H. P. Schwan, K. Li and V. Bryson, "A dielectric
study of the low conductance surface membrane in E. coli,"
Nature, vol. 177, pp. 134-135; 1956.
[8] K. McQuillen, "Bacterial protoplasts," in "The Bacteria," I. C.
Gunsalus and R. Y. Stanier, Eds., Academic Press, Inc., New
York, N.Y., vol. 1, pp. 249-359; 1960.
[9] H. Pauly and H. P. Schwan, "tber die Impedanz einer Sus-
pension von kugelformigen Teilchen mit einer Schale," Z.
Naturforsch., vol. 14 b, pp. 125-131; 1959.
[10] , "The Electrical Conductance and Dielectric Constant of
the Interior of Erythrocytes," University of Pennsylvania,
Electromedical Lab., The Moore School of Elec. Engrg., Phila-
delphia, Pa., ONR Tech. Rept. No. 28; 1959.
[11] H. Pauly, "Electrical conductance and dielectric constant of
the interior of erythrocytes," Nature, vol. 183, pp. 333-334;
1959.
[12] H. Pauly, L. Packer and H. P. Schwan, "Electrical properties
of mitochondrial membranes," J. Biophys. and Biochem. Cytol.,
vol. 7, pp. 589-601; 1960.
[13] H. Pauly and L. Packer, "The relationship of internal con-
ductance and membrane capacity to mitochondrial volume,"
c I-- -0E02 a
J. Biophys. and Biochem. Cytol. vol. 7, pp. 603-612; 1960.
[14] H. P. Schwan, "Electrical properties of tissue and cell sus-
Fig. 7-Plot in the complex admittance and in the complex dielec- pension," in "Advances in Biological and Medical Physics,"
tric plane. Symbols used as in Figs. 4 and 5. J. H. Lawrence and C. A. Tobias, Ed., Academic Press, Inc.,
New York, N.Y., vol. 5, pp. 148-209; 1957.
[15] - , "Biophysics of diathermy," in: "Therapeutic Heat," S.
to 30 atmospheres, which is again three times higher than Licht, Ed., E. Licht, New Haven, Conn., pp. 55-115; 1958.
the value for blood corpuscles with a concentration of [16] H. P. Schwan and K. S. Cole, "Bioelectricity: Alternating
current admittance of cells and tissues," in "Medical Physics,"
small solutes corresponding to an osmotic pressure of 0. Glasser, Ed., Year Book Publisher, Chicago, Ill., vol. 3;
nearly 7 atmospheres. Again, by comparison with the re- 1960.
sults from red blood corpuscles, the mobility of the ions The most recent and comprehensive review about theory and
results of biological impedance measurements is Schwan [141. A re-
within the bacterial cell is roughly reduced by a factor of view about the methods of impedance measurement will be published
2. This is probably due to the electrostatic effect of the by Schwan: "Determination of biological impedances," in: "Physi-
cal Techniques in Biological Research" G. Oster and A. W. Pol-
protein molecules. lister, Eds., Academic Press, Inc., New York, N.Y.; in press.

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