Inuishi, Tsutomu

Muraji, Masafumi

Tsujimoto, Hiroaki

Osaka City University Repository

Mem. Fac. Eng., Osaka City Univ., Vol. 43, pp.13-17 (2002)
On the generating factor of nonlinear dielectric responses
of Sa.ccharomyces cerevisiae
Tsutomu INUISHI *,
Masafumi MURAJI** and Hiroaki TSUJIMOTO ***
(Received October 15, 2002)
Synopsis
The harmonics of response wave based on the nonlinear dielectric properties of yeast cells
have particular patterns for biological activity. The method is a novel technique for
determining the activity of living cells. In this paper, we examined the generating factor of the
nonlinear dielectric responses by comparing the results in the case where yeast cells exist near
the electrodes of an electrochemical cell with those in the case where yeast cells do not. It was
ascertained that nonlinear dielectric responses were induced according to biological activities.
KEYWARDS: Nonlinear dielectric property, Biological activity, Membrane filter, Electrode
1. Introduction
When an ac voltage is applied to cell suspensions and response wave (electric current
wave from cell suspension) is analyzed using FFT, the harmonics of the response wave show
peculiar patterns according to biological activities. It is considered that the electric current
wave is effected by the biological activity of cell. This phenomenon is called nonlinear
dielectric property(l). It is thought that these differences in the responses according to cell
activity are derived from the membrane proteins and are mainly ascribable to by the
H+-ATPase in the plasma membrane.
H. Yositake et a1 studied nonlinear dielectric properties of yeast cells using 2-electrodes
system(2) in which the responses are easier to be influenced by adhesion of yeast cells on
electrodes than 4-electrodes system. We examine generating factor of nonlinear dielectric
responses by using 2-electrodes system. First we measured nonlinear dielectric responses in
each growth phase (2h, 7h, 25h) by using 2-electrode system. Then, to examine the effect of
yeast adhesion on electrode surface, we produced the electrochemical cell to which we can
mount membrane filters. We discuss the generating factor of nonlinear dielectric responses by
comparing the results in the case where yeast cells exist near the electrodes with those in the
case where yeast cells do not.
* Student, Master Course of Department of Physical Electronics and Information
** Lecturer, of Department of Electrical Engineering
***Professor, of Department of Electrical Engineering
13
2. Experimental
2.1 System
The block diagram of the system in this study used for measuring nonlinear dielectric
properties of yeast cells is shown in Fig.1. The sinusoidal waves which were 64 steps per
wavelength were output from a D/A converter in a microcomputer with configured exciting
frequency and field. These waves were applied (14Hz, 1.25V) to the electrochemical cell as the
exciting waves after being smoothed by the analog 2-dimension low pass filter. The .current
waves of the response were logged into the microcomputer with a sampling frequency 32 times
the exciting frequency. The sampled data were FFT analyzed for 512 points per block. As a
result of the nonlinear dielectric properties, the power spectrum was obtained from an average
of 60 blocks. The power spectrum ~ is defined as following equation.
\ :
~ = 20Xlog1o(L-ms/1onnJ [dB] (1)
Here f rn1s is the mea~uredcurrent, forms =1 X 10-10 [A].
PC ~ D/A ~ Low passconverter fi Iter
~ ~
~
AID
..... Op amp f+- Electrochemicalconverter cell
Fig.l. A block diagram of the measurement system
2.2 Electrochemical cell
We produced the electrochemical cell to which we can mount membrane filters as shown in
Fig.2. We compared the nonlinear dielectric properties in the case where yeast suspensions
were filled in all the domains A, Band C with those in the case where suspensions were filled in
the domain B and their supernatant was filled in A and C. The hole size of a membrane filter is
0.45 f..L ill, and yeast (about 5 f..L m) can not pass through membrane filters.
6.3mm
Electrode(Au)
A
ilter I I t
•
•Acrylics
Membrane f
+-+
7 mm
...--.
10.5 mm
Fig.2. Electrochemical cell
14
2.3 Organisms
Yeast cells used in this study were Saccharomyces cereV1Slae DKD - 5DH and were
cultured (main-culture) in 30ml YEPD medium (O.3%yeast extract, O.5%peptone, 1.0%dextrose,
pH5.0) after pre-culture in order to keep the number of yeast always constant. The culture was
maintained at a temperature of 25°C. Fig.3 shows the growth curve of the yeast cells under
these conditions. The measurements were performed in the following growth phases: induction
phase (Cultivation time 2h), logarithmic growth phase (Cultivation time 7h), and stationary
phase (Cultivation time 25h). The· number of cells in each experiment was adjusted to 107ml-1
by centrifugation because the number of cells in suspensions affects to the characteristic
responses(3).
Logarithmic
growth phase
Stationary
phase
28
27
~ 26
"-~ 25
.c
E~ 24
~ 23
..Q
22
21
0 5
Induction
phase
10 15
Time[h]
20 25
Fig.3. Growth curve of Saccharomyces cerevisiae
2.4 Process of measurement
We call the measurement result obtained from cell suspension Sample Data and the result
obtained from its supernatant after centrifugation Control Data. Both are expressed the power
spectrum values shown the equation (1). We regarded the value that was Sample Data minus
Control Data as the result of the nonlinear dielectric properties.
Sample Data
+
Controle Data
Nonlenear dielectric
property
Fig.4. Process for measuring nonlinear dielectric properties
15
3. Result and discussion
Fig.5 shows the electrochemical cell (a) without membrane filter, (b) filled with yeast
suspension in domain A, Band C, and (c) filled with yeast suspension in domain B and filled
with its supernatant in domain A and C, and shows the obtained power spectra of the
nonlinear dielectric properties under each condition. Shadow part in the figure indicates the
domain of yeast suspensions.
Electrode (Au)
(a) ~4
-Ei2
E
to
Q)
0.
V>-2
L.
~4
0
~
-6
Induct i on phase Logar i thm ic growth Stat i onary phase
Membrane filter (2h) phase (7h) (25h)
(b)
~4
Ei
=,2
I-
+oJ
~o
0.
v:>
1--2
Q)
~4
~
-6
Induct i on phase Logar i thll i c growth Stat i onary phase
(2h) phase (7h) (25h)
(C) W{~ ~ 2
"'0
A C ~ 0
I-
+oJ
.. m ~ -2
0.
V')
I- -4
Q)
~-6
~
-H
Induct ion phase Logar i thm i c Stat ionary phase
(2h) growth phase (25h)
(7h)
Fig.5. Result of the nonlinear dielectric properties under the condition
(a)Without membrane filter,
(b)Domain A, B, and C filled with yeast suspension,
(c) Domain B filled with yeast suspension and Domain A and C filled
with its supernatant.
From Fig.5 (a) we can see that the power 'spectra show different pattern according to yeast
activity based on each growth phase. The 3rd harmonic in the induction phase, the 3rd and the
4th harmonic in the logarithmic growth phase and the 3rd harmonic in the stationary phase
16
appeared in the plus side. The 4th and the 5th harmonic in the induction phase and the 5th
harmonic in the logarithmic growth phase, in the minus side. We made experiments to
examine the effect of yeast adhesion on electrodes by using the electrochemical cell mounted
with the membrane filter. We studied the effect of the membrane filter itself on the result of
nonlinear dielectric responses. Cell suspension was put in the domain A, Band C. The graph in
Fig.5 (b) showed that although the data at 2h and 25h had a large change, compared with those
in Fig.5 (a), the data at 7h were relatively small. It follows from this that when yeast cells are
relatively non-active in the induction phase and the stationary phase, the power spectra are
influenced by the membrane filter as well as by biological activity, and when yeast cell is very
active in the logarithmic growth phase, nonlinear responses by the biological activity of yeast
cells are more dominant than by the membrane filter itself. In Fig.5 (c) the harmonics in the
induction phase and the stationary phase hardly appeared. The 2nd and the 4th harmonics in
the logarithmic growth phase appeared in the minus side greatly. Since the results of the
nonlinear dielectric responses are Sample Data (power spectrum of yeast suspension) minus
Control Data (power spectrum of its supernatant), it can be said that at 2h and 25h the power
spectrum of Sample Data was almost the same as the power spectrum of the Control Data.
Therefore we can say that, at 2h and 25h, the supernatant in domain A and C strongly
reflected to response results and the effect of yeast suspensions in domain B are small. On the
other hand, it is considered that yeast cells in the domain B at 7h which have strong
multiplication capability and activity reflect to response results strongly rather than the effect
of the supernatant near electrodes.
In concluding, we can say that nonlinear dielectric responses were induced according to
biological activities based on each growth phase. Therefore this method can be useful to acquire
the biological activities of yeast cells.
Reference
(1) A.M.Woodward, D.B.Kell: "On the nonlinear dielectric properties of biological systems
Saccharomyces cerecisiae" ,Bioelectrochemistry and Bioenergetics, 24,pp.83-100 (1990)
(2) H.Yositake, M.Muraji, A.Tsujimoto, W.Tatebe: "The estimation of the yeast growth
phase by nonlinear dielectric properties of the measured electric current" ,Journal of
Electroanalytical Chemistry, 496, pp.148-152 (2001)
(3)H.Yositake, M.Muraji, A.Tsujimoto, W.Tatebe: "The changing of nonlinear dielectric portieres
for cultured yeast cells in each growth phase" , lEE of Japan A,120, pp.142 (2000)
17


On the generating factor of nonlinea dielectric responses of Saccharomyces cerevisiae

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On the generating factor of nonlinea dielectric responses of Saccharomyces cerevisiae

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