International Journal of Inorganic Materials 3 (2001) 643646

Influence of particle size on the antibacterial activity of zinc oxide

Osamu Yamamoto*
Department of Applied Chemistry, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi 243 -0292, Japan
Received 3 April 2000; accepted 14 August 2001

Abstract
The influence of particle size on the antibacterial activity of ZnO powders was investigated using powders with different particle sizes
ranging from 0.1 to 0.8 mm. By measuring the change in electrical conductivity with bacterial growth, it was found that the antibacterial
activity of ZnO increased with decreasing particle size and increasing powder concentration. The changes of antibacterial action for
Staphylococcus aureus were similar to those for Escherichia coli. However, the influence of particle size for Staphylococcus aureus was
less than that for Escherichia coli. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Zinc oxide; Powder; Antibacterial activity

1. Introduction
Microbial pollution and contamination by microorganisms have produced various problems in industry and
other vital fields, such as degradation and infection. In
order to solve these problems, new pasteurization and
antibacterial techniques have been demanded and studied
[13].
The antibacterial activity of ceramic powders has attracted attention as a new technique that can substitute for
conventional methods using organic agents. Ceramic powders of zinc oxide (ZnO), calcium oxide (CaO) and
magnesium oxide (MgO) were found to show marked
antibacterial activity [413]. The use of these ceramics has
the following advantages: they contain mineral elements
essential to humans and exhibit strong antibacterial activity
in small amounts without the presence of light. It was
found that ZnO exhibits antibacterial activity at pH values
in the range from 7 to 8 [4], and these values are suitable
for use in water used for washing and drinking. The
antibacterial activity of ZnO is considered to be due to the
generation of hydrogen peroxide (H 2 O 2 ) from its surface
[14]. However, it is not yet clear what changes in
antibacterial activity are expected due to the particle size
of ZnO.
In the present work, ZnO powders with different particle
*Tel.: 181-46-291-3148; fax: 181-46-242-8760.
E-mail address: yamamoto@chem.kanagawa-it.ac.jp (O. Yamamoto).

sizes were prepared by crushing ZnO heated at 14008C.

After preparing slurries of the powders, the change in
antibacterial activity as a function of particle size was
studied by measuring the change in electrical conductivity
with bacterial growth.

2. Experimental

2.1. Preparation of powder samples

Reagent grade ZnO powder was used as starting material. The powder, with a particle size of about 0.8 mm, was
heated at 14008C for 3 h in air and then milled using a
planetary ball mill. The sample code, the particle size and
the specific surface area of the powder samples obtained
are listed in Table 1. The powder samples were suspended
in physiological saline in the concentration range from 0.4
Table 1
Sample code, particle size and specific surface area of the ZnO powders
used in this study
Sample
code

Particle
size (mm)

Specific surface
area (m 2 g 21 )

ZO-1
ZO-2
ZO-3
ZO-4
ZO-5

0.1
0.2
0.3
0.5
0.8

26.0
23.8
11.1
2.11
0.85

1466-6049 / 01 / $ see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S1466-6049( 01 )00197-0

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O. Yamamoto / International Journal of Inorganic Materials 3 (2001) 643 646

3. Results

Fig. 1. Schematic illustration of the apparatus used in the antibacterial

tests.

to 100 mg cm 23 and then the prepared slurries were used

in antibacterial tests.
The particle size of the ZnO powders was examined
using a scanning electron microscope (SEM, JXA840).
The specific surface area of the powders was measured by
an OMNSORP 100CX model.

2.2. Preparation of bacterial suspensions

The specific surface areas of the powder samples

increased with decreasing particle size, that is, the value
increased in the range from 0.85 to 26.0 m 2 g 21 . From
XRD measurements, the diffraction peaks corresponding to
hexagonal-type ZnO appeared in all powder samples.
Regarding growth of the bacteria, it is known that
electrolytes such as organic and amino acids are produced
with the digestion of proteins in the medium [15]. The
electrical conductivity in such a growth medium, therefore,
increases with increasing amount of electrolytes produced,
the change occurring at a bacterial concentration of about
10 7 CFU cm 23 in the medium.
Fig. 2a and b show the changes in electrical conductivity
with incubation time for E. coli and S. aureus, respectively, ZO-5 being used. DT (detection time) indicates the
incubation time at which an electrical change can be
detected. Hence, if the value of DT is delayed by adding
the powder samples, it can be concluded that the samples
have the effect of inhibiting bacterial growth. In the case
where no ZnO powder was added (control), the DT value
for E. coli was approximately 7 h. On adding ZO-5,
however, the DT value increased with increasing powder

Staphylococcus aureus 9779 (hereafter, S. aureus) and

Escherichia coli 745 (E. coli) were used as the test
bacteria. The bacteria were cultured in Brain Heat Infusion
(BHI) at 378C for 24 h on a reciprocal shaker. The
bacterial culture was suspended in sterile physiological
saline at a final concentration of approximately 10 2 CFU
cm 23 (CFU: Colony Forming Unit).

2.3. Tests of antibacterial activity

The antibacterial activity of the powder samples was
assessed by measuring the change in electrical conductivity
with bacterial growth. The apparatus for measuring the
conductivity was a Bactometer Microbial Monitoring
System Model 64 (bioMerieux), as shown in Fig. 1.
Placing the bacteria into the wells of a module of the
Bactometer was carried out as follows: the powder samples
were placed in a well containing Modified Plate Count
Agar (MPCA) and then the bacterium suspension was
dispensed into the well. After setting the module in the
Bactometer, the change in electrical conductivity was
monitored during incubation at 37 o C for 25 h in the
absence of light. Details of the procedures were reported in
previous publications [5,1013].
In order to determine indirectly the pH when the powder
samples were added to the well, the samples were dispersed in physiological saline at a powder concentration of
6.4 mg cm 23 . After allowing the dispersed solutions to
stand for 24 h, the pH of the physiological saline was
measured.

Fig. 2. Changes in electrical conductivity with incubation time for (a) E.

coli and (b) S. aureus.

O. Yamamoto / International Journal of Inorganic Materials 3 (2001) 643 646

concentration and no DT value could be detected at a

powder concentration of 50 mg cm 23 (see Fig. 2a). The
change in the DT value for S. aureus was similar to that

645

for E. coli, and no DT was observed at a powder

concentration of 1.6 mg cm 23 (see Fig. 2b). The results
indicate an increase in antibacterial activity on increasing
the concentration of the powder in the medium.
Based on the change in electrical conductivity described
above, the antibacterial activity of all powder samples was
examined for two bacteria, E. coli and S. aureus.
Fig. 3a and b compare the antibacterial activity of five
powder samples towards E. coli and S. aureus, respectively. The vertical axis, DT / DT cont. , represents the ratio of
the DT values at specified powder sample concentrations
to that for no powder sample addition (control). If the
values of DT / DT cont. change with a steep rise at lower
powder concentrations, it can be taken to show stronger
antibacterial activity. As shown in Fig. 3a, with increasing
particle size of the ZnO powder, a pronounced change in
the value was observed at high powder concentrations, that
is, a decrease in the powder particle size resulted in
effective antibacterial activity with respect to E. coli. For
S. aureus (see Fig. 3b), a change in the DT / DT cont. value
occurred at a slightly lower powder concentration with
increasing powder particle size than for E. coli. The
changes in antibacterial activity of the powder samples
with respect to S. aureus were similar to those for E. coli,
but the effect of particle size on the antibacterial activity
for S. aureus was less than that for E. coli.
The pH of the powder samples dispersed in physiological saline was 7.5 for all samples.

4. Discussion

Fig. 3. Comparison of the antibacterial activity of the powder samples

with respect to (a) E. coli and (b) S. aureus: (n) ZO-1, (h) ZO-2, (s)
ZO-3, (,) ZO-4, (d) ZO-5.

By measuring the changes in electrical conductivity with

bacterial growth, it was found that the antibacterial activity
increased with decreasing particle size of the ZnO power.
The following four factors may affect the antibacterial
activity of ceramic powders: (1) the cations eluted from
the powder, (2) active oxygen generated from the powder,
(3) the pH, and (4) mechanical destruction of the cell
membrane [46,1013]. However, Yamamoto et al. [4,13]
and Sawai et al. [14] reported that factors (1) and (4) had
no effect on the activity. The pH of the powder samples
dispersed in physiological saline was 7.5, irrespective of
the particle size of the sample. However, this generally
does not affect bacterial growth [16,17]. For the antibacterial activity of ZnO, Yamamoto et al. reported the
generation of hydrogen peroxide, H 2 O 2 , from the surface
of ZnO and considered this to be effective for the
inhibition of bacterial growth [4]. It can be assumed that
the concentration of H 2 O 2 generated from the surface
increases with decreasing particle size, because the number
of ZnO powder particles per unit volume of powder slurry
increases with decreasing particle size. Based on the
above, the increase in antibacterial activity is assumed to

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O. Yamamoto / International Journal of Inorganic Materials 3 (2001) 643 646

be due to the increase in H 2 O 2 generated from the surface

of ZnO on reducing the particle size of the powder
samples.
For ZnO powders, the influence of particle size on S.
aureus was less than that on E. coli. The structures and
chemical compositions of the cell surface of the bacteria
used in this study are quite different. Thin layers of lipid
A, lipopolysaccharide and peptidoglycan are present on the
cell surface of E. coli, whereas there is only a peptidoglycan layer for S. aureus. Sawai et al. carried out an
experiment to determine whether or not the H 2 O 2 generated from ZnO was related to the antibacterial activity by
using four kinds of antibiotics [14]. In the investigation,
the changes in sensitivity of E. coli to the antibiotics
suggested that H 2 O 2 was one of the primary factors
contributing to the antibacterial activity of ZnO. Saito et al.
reported that the H 2 O 2 generated can readily penetrate the
cell wall of the bacteria [18]. Therefore, the differences in
antibacterial action towards S. aureus and E. coli are
assumed to be due to the different sensitivities towards
H2O2.

5. Conclusion
The changes in antibacterial activity of ZnO powders
with different particle sizes were studied. The antibacterial
activity of ZnO powder increased with decreasing particle
size and increasing powder concentration. The changes in
antibacterial action towards S. aureus were similar to those
for E. coli. However, the influence of particle size on
antibacterial activity towards S. aureus was less than that
for E. coli. The occurrence of antibacterial activity was
assumed to be due to the generation of H 2 O 2 from the
surface of ZnO.

Acknowledgements
The present work was partly supported by a Grant-inAid for Scientific Research (C) (No. 12650676) from the
Japan Society for the Promotion of Science.

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