1. JOURNAL01~I~RMJBNTATIONANDBIO~NO~O
Vol. 75, NO. 2, 99-102. 1993
Resistance of Yeast and Bacterial Spores to High Voltage Electric Pulses
YOSHIMASA YONEMOTO, l TETSUO YAMASHITA, 1MASAFUMI MURAJI, 2 WATARU TATEBE,~-
HIROSHI OOSHIMA, 3 JYOJI KATO,3AKIRA KIMURA,4 AND KOUSAKU MURATA4.
Division of Food &BeverageResearch, Otsuka Chemical Co., Ltd., Kawauchi, Tokushima 771-01,1Department
of ElectricalEngineering,2Department of Bioapplied Chemistry,3Osaka City University, Sugimoto,
Sumiyoshi-ku, Osaka 558, and Research Institute for Food Science, Kyoto
University, Ufi, Kyoto 611,4Japan
Received8 September1992/Accepted19November1992
Spores of a yeast, Saccharomycescerevisiae, and a bacterium, BacillussubtUis, were exposed to high voltage
electric pulses. The vinbillties of spores and vegetative cells of the yeast were significantly decreased after the
electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phlox-
ine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little
decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results
revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.
As described previously (1), microbial spores are excep-
tionally resistant to extreme environments such as high tem-
perature, high osmotic pressure, high and low pHs, toxic
compounds and mechanical shocks compared with vegeta-
tive cells, although they have proteins (enzymes), nucleic
acids, membranes and structures basically similar to those
of vegetative cells. Our recent studies also indicated that
yeast spores contain all of the enzymes present in vegeta-
tive cells at almost the same activity levels (2), and that
bacterial (endo)spores are highly resistant to lytic en-
zymes produced by microbes unless they are physically
damaged (1).
These structural and biochemical properties of micro-
bial spores suggest that they can be used as a biocatalyst
in place of vegetative cells, and in fact we have already
used yeast spores as a biocatalyst for the continuous dephos-
phorylation of p-nitrophenylphosphate in a bioreactor
system (3). In order to use the various enzymes in micro-
bial spores, efficient methods of inducing enzyme activ-
ities in the spores must be developed. A few reports on
the action of electric pulses on the viability and perme-
ability of vegetative ceils of yeast and bacteria have been
published (4, 5). An analysis of the effect of electric pulses
revealed that they caused reversible loss of permeability
(5, 6); this phenomenon has been utilized for the intro-
duction of genetic materials into microbial cells (7, 8).
However, the effect of electric pulses on microbial spores
has not been investigated thus far. As a first step towards
the preparation of enzymatically active microbial spores,
we investigated the effect of electric pulses on the structure
and viability of yeast and bacterial spores.
MATERIALS AND METHODS
Slmres and vegetative cells Cells of a yeast, Saccha-
romyces cerevisiae 4011, and a bacterium, Bacillus subtilis
no. 1 (1), were aerobically grown at 30°C for 20h in
100 ml of YPD (2.0% glucose, 1.0% bactopeptone, 0.5%
yeast extracts: pH 5.0) and NS (0.5% meat extracts, 1.0%
peptone, 0.5% NaCI: pH 7.2) media, respectively. Bac-
topeptone and yeast extracts were purchased from Difco
* Correspondingauthor.
Laboratories, Detroit, Michigan, USA. The cells (vegeta-
tive cells) were collected, washed once in chilled 0.85%
NaC1 and then suspended in the same solution to make
a concentration of 3-5 x l0s cells/ml. Spores of the two
strains were prepared by the same procedures as those
described previously (1, 3). Spores were also suspended
in 0.85% NaCI to give a concentration of approximately
l0s spores/ml. Yeast spores form aggregates in aqueous con-
ditions; the yeast spore suspension was thus vigorously
stirred before use.
Electric pulses The electric circuit used for the pulse
treatment of vegetative cells and spores is illustrated in
Fig. 1. The cell or spore suspension (2.0 ml) was placed
between the two electrodes in the chamber and pulsed 3
or 10 times for 90 ps each time with an interval of 30 s
between pulses. These conditions are nearly the same as
those used for the transformation of yeast cells (8). The
detailed pulse conditions are described under Results and
Discussion.
Viability Vegetativecells and spores before the after
treatment with electric pulses were diluted with 0.85%
NaC1 and spread on agar (1.5%) plates of YPD (for the
lI
01110 /
$1 ~ %-
55 mm
DCPowerCondenser
Source S itF /
/
Chamber
99
FIG. 1. Electriccircuit for electroporation. The conditions for
the pulseexperimentsare describedin the text. S1, Switchfor charg-
ing condenser;$2, switchfor dischargeto chamber.

2. 100 YONEMOTOET AL. J. FEINT. BXO~G.,
yeast) and NS (for the bacterium) media. Plates were incu-
bated at 30°C and colonies on the plates were counted.
Phloxine B uptake In order to discern any changes
in the permeabilities of vegetative cells and spores of the
yeast after the electric pulse, 20/~1 of 10 mg/ml phloxine B
(Sigma Chemical Co., St. Louis, MO, USA) dissolved in
0.85% NaCI was added to 0.2 ml of spore or vegetative cell
suspension before and exactly 1 rain after the electric pulse
treatment (8). After incubation at room temperature for
2 min, the number of spores or cells stained with the red
dye agent was counted by using an optical microscope.
Micrographs Yeast spores were treated with 2.0%
glutaraldehyde for 1 h at room temperature, dried on a crit-
ical dryer (Hitachi CTD-1) and then examined by scanning
electron microscope (Hitachi S-450). Yeast spores after in-
cubation with phloxine B were directly examined as above.
Bacterial spores were negatively stained with phospho-
tungstic acid in 2% KOH, and an electron micrograph
was taken by a transmission electron microscope (Hitachi
H-700).
RESULTS AND DISCUSSION
Electric pulse treatment The electric circuit for the
pulse experiments is shown in Fig. 1. Using electrical
energy from a dc power source the condenser (capacitance,
C=8 pF) was first charged by closing S1. The energy was
then discharged to the vegetative cell or spore suspension,
which was placed between the two electrodes in the cham-
ber, by opening S1 and closing $2. The waveforms of the
voltage between the electrodes (Fig. 2A) and the current
through the suspension (Fig. 2B) were observed on a dig-
ital storage oscilloscope (DCS-9300 Kenwood). The initial
field strength was calculated to be 5,400 V/cm, since the
distance between the two electrodes was 0.5 cm (Fig. 1). If
a suspension contains components of resistance R and the
circuit is ideally regarded as an RC series circuit, the volt-
age V between the two electrodes after the circuit is closed
is expressed as:
(v)
3000
2000
1000
0
(A)
300
200
100
0
~ 9 0 p s
(A)Vo~age
(13)Current
FIG. 2.
sions.
I i I , I , I
0 100 200 300 (its)
Voltage (A) and current (B) curves applied to suspen-
V= Vo exp (- t/r),
Where r is the time constant (r =RC) that indicates the
period of an electric pulse applied to the suspension. The
time constant r was determined to be 90 ps by approximat-
ing the voltage waveform with the least square method to
an ideal exponential damped wave through Vo at t=0. In
order to elucidate the behaviour of the yeast and bacterial
spores toward high voltage electric pulses, 90 ps pulses
with voltage and current waveforms as shown in Fig. 2
were applied to the spore and vegetative suspensions, with
an interval of 30 s between pulses.
Effect of electric pulse on yeast spores Spores and
vegetative cells of S. cerevisiae 4011 were highly suscepti-
ble to the high voltage electric pulse treatment, their viabil-
ities being greatly reduced (more than 90°/~) after pulses
were repeated ten times (Fig. 3A). The spores before and
after electric pulse treatment were examined by scanning
electron microscope (Figs. 4A-1 and A-2). Little structural
change was observed between the two spore preparations.
However, spores with many small holes on their surfaces
were found only in the spore population after the electric
pulse treatment (Fig. 4A-2, indicated by an arrow); the fre-
quency of appearance of such spores was approximately
0.1% under the conditions employed. Although no direct
I00 I00
K
5o 5o .=_
X
_o
e=
0
h.=
"- 0
•-= too>
.g
L..
.=,
50
O
3 I0
]Pulses (times)
FIG. 3. Effectof electric pulses on vegetative cells and spores.
(A) Effect on S. cereviaiae. Vegetativecells and spores were dectri-
caliypulsed bythe method describedin text. Viabilityand phloxineB
uptake are expressed as a function of the number of times electric
pulses applied to the suspension. Phloxine B uptake represents the
percentage of spores or vegetative cells stained with the agent. ),
Viabilityof spores; O, viabilityof vegetativecells; m, phloxineBup-
take by spores; ~3,phloxineBuptake byvegetativecells.(B)Effecton
B. subtilis. Viabilityof vegetativecellsand spores after electricpulses
was determined and expressedas above. ), Viability of spores; o,
viability of vegetativecells.

3. VoL.75, 1993 RESISTANCEOF MICROBIALSPORESTO ELECTRIC PULSES 101
A
1 2
B
FIG. 4. Effectof electricpulseson structure and uptake of phloxineB of yeastspores. (A) Scanningelectronmicrographsof yeastspores
before [1]and after [2]treatmentwithelectricpulsesthreetimes.A beehive-likesporeis indicatedby an arrow. Barsrepresent1.0pm in length.
(B) Photographs of spores stained withphloxineB before [1] and after [2] treatment withelectricpulsesthree times. Arrows in B2 indicate
sporesstained red by phloxineB. Barsrepresent6.0/an in length.
evidence was obtained, such beehive-like spores seemed to
be one of the products of the high voltage electric pulse
treatment.
Vegetative cells after the electric pulse treatment were
efficientlystained with phloxine B, an agent often used for
biostaining (8) (data not shown); the number of cells
stained with the agent increased proportionally with the
increase in the non-viable cell number (Fig. 3A), thus indi-
caring that phloxine B could penetrate only into non-
viable cells. Yeast spores after the electric pulse treatment
were also found to be stained red in the presence of phlox-
ine B (Fig. 4B-2). However, the number of spores stained
with the agent was not proportional to that of non-viable
spores (Fig. 3A). Judging from the size of spores presented
in the scanning electron micrograph (Fig. 4A), the spore
preparation used in this study contained no vegetative
cells. Therefore, the non-proportional result observed on
the numbers of non-viable and phloxine B-stained spores
was presumably due to differential damage done to the
spores by the electric pulses, since yeast spores have a ten-
dency to form aggregates in aqueous solutions and despite
vigorous stirring before use the aggregations were not
eliminated, even after the electric pulse treatment (data
not shown). The pldoxine B uptake results indicate that
the electric pulse method can render spores, as well as
vegetative cells, permeable to some chemicals and that
this method may be applicable to the preparation of enzy-
matically active spores for use as a biocatalyst. The activa-
tion of spores by this method and other physicochemical
approaches will be reported elsewhere.
Effect of electric pulse on bacterial spores Spores
and vegetative cells of B. subtilis no. 1 were also treated by
electric pulses (Fig. 3B). Contrary to the case of the yeast
S. cerevisiae (Fig. 3A), the bacterial spores were highly
resistant to the treatment and Httle decrease in viability
was observed, although vegetative cells sharply lowered
their viability. Electron microscopic observation of spores
before and after the electric pulse treatment revealed
that they were structurally indistinguishable from each
other. However, spores after the treatment had cracks on
their surfaces (Fig. 5B-2, indicated by arrows) and black
granules initially contained in the spores as 2-3 particles
(Fig. 5A-2) were crushed, increasing in numbers. When the
bacterial spores were ultrasonically treated, the granules
were completely shattered into many small particles with
a concomitant decrease in viability (1). Therefore, the
repeated application of electric pulses may cause a de-
crease in the viability of bacterial spores.
Thus, the high voltage electric pulse experiments indi-
cated that bacterial (endo) spores are highly resistant to
physical shocks and are structurally and/or biochemically
different from yeast spores. We have obtained enzyme(s)

4. 102 YONEMOTO ET AL. J. FERMENT.BIOENG.,
! 2
FIG. 5. Effectof electric pulses on structure of bacterial spores. Electron micrographs of spores were taken before (A) and after (B)
treatment with electricpulse three times. One of the spores in AI and BI is magnifiedand shown in the right panel. Arrows in B2 indicate cracks
formed after electric pulses. Bars represent 1.0/~min length.
that induce lysis in bacterial spores (1). Recently, we have
also found a bacterium producing enzyme(s) responsible
for the lysis of yeast spores (unpublished data). The use of
these enzymes that lyse yeast and bacterial spores may facil-
itate a structural comparison of prokaryotic and eukary-
otic spores.
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