Influence of Particle Size on the Antibacterial Activity

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International Journal of Inorganic Materials 3 (2001) 643646

Influence of particle size on the antibacterial activity of zinc oxide

*Osamu YamamotoDepartment 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 forStaphylococcus 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 sizes were prepared by crushing ZnO heated at 14008C.

After preparing slurries of the powders, the change in

Microbial pollution and contamination by microorga- antibacterial activity as a function of particle size was

nisms have produced various problems in industry and studied by measuring the change in electrical conductivity

other vital fields, such as degradation and infection. In with bacterial growth.

order to solve these problems, new pasteurization andantibacterial techniques have been demanded and studied

[13]. 2. Experimental

The antibacterial activity of ceramic powders has at-

tracted attention as a new technique that can substitute for 2.1. Preparation of powder samples

conventional methods using organic agents. Ceramic pow-

ders of zinc oxide (ZnO), calcium oxide (CaO) and Reagent grade ZnO powder was used as starting materi-

magnesium oxide (MgO) were found to show marked al. The powder, with a particle size of about 0.8mm, was

antibacterial activity [413]. The use of these ceramics has heated at 14008C for 3 h in air and then milled using a

the following advantages: they contain mineral elements planetary ball mill. The sample code, the particle size and

essential to humans and exhibit strong antibacterial activity the specific surface area of the powder samples obtained

in small amounts without the presence of light. It was are listed in Table 1. The powder samples were suspended

found that ZnO exhibits antibacterial activity at pH values in physiological saline in the concentration range from 0.4in the range from 7 to 8 [4], and these values are suitable

for use in water used for washing and drinking. The Table 1antibacterial activity of ZnO is considered to be due to the Sample code, particle size and specific surface area of the ZnO powders

used in this studygeneration of hydrogen peroxide (H O ) from its surface2 2

[14]. However, it is not yet clear what changes in Sample Particle Specific surface2 21antibacterial activity are expected due to the particle size code size (mm) area (m g )

of ZnO. ZO-1 0.1 26.0In the present work, ZnO powders with different particle ZO-2 0.2 23.8

ZO-3 0.3 11.1

ZO-4 0.5 2.11*Tel.: 181-46-291-3148; fax: 181-46-242-8760.

ZO-5 0.8 0.85E-mail address: [email protected] (O. Yamamoto).

1466-6049/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved.

P II: S 1 4 6 6 -6 0 4 9 (0 1 )0 0 1 9 7 -0


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644 O. Yamamoto /International Journal of Inorganic Materials 3 (2001) 643646

3. Results

The specific surface areas of the powder samples

increased with decreasing particle size, that is, the value2 21

increased in the range from 0.85 to 26.0 m g . From

XRD measurements, the diffraction peaks corresponding to

hexagonal-type ZnO appeared in all powder samples.

Regarding growth of the bacteria, it is known thatelectrolytes 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,Fig. 1. Schematic illustration of the apparatus used in the antibacterialtests. increases with increasing amount of electrolytes produced,

the change occurring at a bacterial concentration of about7 23

10 CFU cm in the medium.23

to 100 mg cm and then the prepared slurries were used Fig. 2a and b show the changes in electrical conductivity

in antibacterial tests. with incubation time for E. coli and S. aureus, respective-

The particle size of the ZnO powders was examined ly, ZO-5 being used. DT (detection time) indicates the

using a scanning electron microscope (SEM, JXA840). incubation time at which an electrical change can be

The specific surface area of the powders was measured by detected. Hence, if the value of DT is delayed by adding

an OMNSORP 100CX model. 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

2.2. Preparation of bacterial suspensions 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 physiological2

saline at a final concentration of approximately 10 CFU23

cm (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 waso

monitored during incubation at 37 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 dis-

persed in physiological saline at a powder concentration of23

6.4 mg cm . After allowing the dispersed solutions to

stand for 24 h, the pH of the physiological saline was Fig. 2. Changes in electrical conductivity with incubation time for (a)E.measured. coli and (b) S. aureus.


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O. Yamamoto /International Journal of Inorganic Materials 3 (2001) 643646 645

concentration and no DT value could be detected at a for E. coli, and no DT was observed at a powder23 23

powder concentration of 50 mg cm (see Fig. 2a). The concentration of 1.6 mg cm (see Fig. 2b). The results

change in the DT value for S. aureus was similar to that 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 fivepowder samples towards E. coli and S. aureus, respective-

ly. The vertical axis, DT/ DT , represents the ratio ofcont.

the DT values at specified powder sample concentrations

to that for no powder sample addition (control). If the

values of DT/DT change with a steep rise at lowercont.

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 valuecont.

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 physiologi-

cal saline was 7.5 for all samples.

4. Discussion

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 anti-

bacterial activity of ZnO, Yamamoto et al. reported the

generation of hydrogen peroxide, H O , from the surface2 2

of ZnO and considered this to be effective for the

inhibition of bacterial growth [4]. It can be assumed that

the concentration of H O generated from the surface2 2

increases with decreasing particle size, because the number

of ZnO powder particles per unit volume of powder slurryFig. 3. Comparison of the antibacterial activity of the powder samples

increases with decreasing particle size. Based on thewith respect to (a) E. coli and (b) S. aureus: (n) ZO-1, (h) ZO-2, (s)ZO-3, (,) ZO-4, (d) ZO-5. above, the increase in antibacterial activity is assumed to


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646 O. Yamamoto /International Journal of Inorganic Materials 3 (2001) 643646

be due to the increase in H O generated from the surface Acknowledgements2 2

of ZnO on reducing the particle size of the powder

samples. The present work was partly supported by a Grant-in-

For ZnO powders, the influence of particle size on S. Aid for Scientific Research (C) (No. 12650676) from the

aureus was less than that on E. coli. The structures and Japan Society for the Promotion of Science.

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 thecell surface of E. coli, whereas there is only a peptido-

Referencesglycan layer for S. aureus. Sawai et al. carried out an

experiment to determine whether or not the H O gener-2 2

[1] Kusaka T, Takagi Y. J Antibact Antifungal Agents 1992;20:451.ated from ZnO was related to the antibacterial activity by

[2] Saito M. J Antibact Antifungal Agents 1993;21:17.using four kinds of antibiotics [14]. In the investigation, [3] Tsunoda Y, Egawa H, Yuge O. J Antibact Antifungal Agentsthe changes in sensitivity of E. coli to the antibiotics 1992;20:571.

[4] Yamamoto O, Hotta M, Sawai J, Sasamoto T, Kojima H. J Ceramsuggested that H O was one of the primary factors2 2

Soc Jpn 1998;106:1007.contributing to the antibacterial activity of ZnO. Saito et al.[5] Yamamoto O, Sawai J, Sasamoto T. J Inorg Mater 2000;2:451.

reported that the H O generated can readily penetrate the2 2 [6] Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem

cell wall of the bacteria [18]. Therefore, the differences in Eng Jpn 1995;28:288.antibacterial action towards S. aureus and E. coli are [7] Sawai J, Kawada E, Kanou F, Igarashi H, Hashimoto A, Kokugan T

et al. J Chem Eng Jpn 1996;29:251.assumed to be due to the different sensitivities towards[8] Yamamoto T, Uchida M, Kurihara Y. J Antibact Antifungal AgentsH O .

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[9] Kurihara Y. New Ceram 1996;1996:39.

[10] Yamamoto O, Sawai J, Hotta M, Kojima H, Sasamoto T. J Mater5. Conclusion Sci Soc Jpn 1998;35:258.

[11] Sawai J, Yamamoto O, Hotta M, Kojima H, Sasamoto T. J Chem

Soc Jpn 1998;1998:633.The changes in antibacterial activity of ZnO powders[12] Yamamoto O, Sawai J, Ishimura M, Kojima H, Sasamoto T. Jwith different particle sizes were studied. The antibacterial

Ceram Soc Jpn 1999;107:853.activity of ZnO powder increased with decreasing particle [13] Yamamoto O, Shimura T, Sawai J, Kojima H, Sasamoto T. J Ceramsize and increasing powder concentration. The changes in Soc Jpn 2000;108:156.

[14] Sawai J, Kojima H, Igarashi H, Hashimoto A, Shoji S, Kokugan T etantibacterial action towards S. aureus were similar to thoseal. J Ferment Bioeng 1998;86:521.for E. coli. However, the influence of particle size on

[15] Firstenberg-Eden R, Eden G. In: Impedance microbiology, Letch-antibacterial activity towards S. aureus was less than thatworth: Research Studies Press, 1984, p. 7.

for E. coli. The occurrence of antibacterial activity was [16] Radford SA, Board RG. Appl Microbiol 1995;20:11.assumed to be due to the generation of H O from the [17] Lorence J. Manuf Chem 1998;69:22.2 2surface of ZnO. [18] Saito I, Matsuno S. Proteins, Nucleic Acids Enzymes 1988;33:266.

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Influence of Particle Size on the Antibacterial Activity