African Journal of Microbiology Research Vol. 5(12), pp. 1368-1373, 18 June, 2011 [623164]

African Journal of Microbiology Research Vol. 5(12), pp. 1368-1373, 18 June, 2011
Available online http://www.academicjournals.org/ajmr
DOI: 10.5897/AJMR10.159
ISSN 1996-0808 ©2011 Academic Journals

Full Length Research Paper

Antibacterial activity of ZnO nanoparticle on gram-
positive and gram-negative bacteria

Zarrindokht Emami-Karvani* and Pegah Chehrazi

Islamic Azad University, Falavarjan Branch, Iran.

Accepted 10 May, 2011

The aim of the present study is to determine the antimicrobial activity of ZnO nanoparticles against
Gram-negative and Gram-positive bacteria. Escherichia coli (E. coli) and Staphylococcus aureus (S.
aureus) were used as test microorganisms. The effects of particle size and concentration on the
antibacterial activity of ZnO nanoparticles was studied using bacteriological tests such as disc and well
diffusion agar methods, minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC). These tests were performed in nutrient broth and nutrient agar following standard
methods. In addition, the effect of different concentrations of ZnO nanoparticles on the growth of E. coli
and S. aureus was measured with respect of time. The minimum inhibitory concentration was
determined using seven different concentrations of ZnO nanoparticles including 16, 8, 4, 2, 1 and 0.5
mg/ml. The MIC value for E. coli and S. aureus was 1 and 0.5 mg/ml, respectively. The results showed
that ZnO nanoparticles have antibacterial inhibition zone of 29 and 19 mm at the concentration of 10
mg/ml against E. coli and S. aureus, respectively. Gram-negative bacteria seemed to be more resistant
to ZnO nanoparticles than Gram-positive bacteria. It was found that the antibacterial activity of ZnO
nanoparticles increased with decreasing particle size and increasing powder concentration. The
antibacterial effect of ZnO nanoparticles was time dependent and takes effect gradually. ZnO bulk
powder showed no significant antibacterial activity.

Key word: ZnO nanoparticle, Escherichia coli, Staphylococcus aureus, minimum inhibitory concentration (MIC),
minimum bactericidal concentration (MBC).

INTRODUCTION

Nanoparticlesare is a special group of materials with
unique features and extensive applications in diverse
fields (Matei et al., 2008). Studying these particular
features has always been of great interest to many
scientists. In fact, nanoparticles display completely
unique properties in comparison with their bulk size
counterparts (Priyanka et al., 2009). A large number of
materials which were considered to be safe develop
toxicity at nano size ranges (Reddy et al., 2007) which is
mainly related to the increased specific surface area and
high reactivity of nano size materials (Nagarajan and

*Corresponding author. E-mail: [anonimizat],
[anonimizat]. Tel: +98-913-2034159. Fax: +98-312-
3120136. Rajagopalan, 2008; Laura et al., 2006). A larger surface
area (as in case of nanoparticles) ensures an increased
range of probable interaction with bio-organics present on
the viable cell surface (Rizwan et al., 2010c). The
considerable antimicrobial activities of inorganic metal
oxide nanoparticles such as ZnO, MgO, TiO2, SiO2 and
their selective toxicity to biological systems suggest their
potential application as therapeutics, diagnostics,
surgicaldevices and nanomedicine based antimicrobial
agents (Mohsen and Zahra, 2008; Sobha et al., 2010;
Laura et al., 2006; Sawai and Yoshikawa, 2003; Reddy et
al., 2007). The advantages of using these inorganic
oxides nanoparticles as antimicrobial agents are their
greater effectiveness on resistant strains of microbial
pathogens, less toxicity and heat resistance. In addition,
they provide mineral elements essential to human cells
and even small amounts of them exhibit strong activity

(Nagarajan and Rajagopalan, 2008; Toshiaki et al., 2008;
Zakaria et al., 2010)
Among metal oxide nanoparticles,
ZnO nanoparticles as one of the multifunctional inorganic
nanoparticles has many significant features such as
chemical and physical stability, high catalysis activity,
effective antibacterial activity as well as intensive
ultraviolet and infrared adsorption with broad range of
applications as semiconductors, sensors, transparent
electrodes, solar cells, etc. (Matei et al., 2008; Kalyani et
al., 2006). Also in recent years ZnO has received
considerable attention because of its unique optical,
piezoelectric, and magnetic properties (Marcus and Paul,
2007). In addition ZnO nanoparticles has the potential to
impact many aspects of food and agricultural systems
because of its antimicrobial efficacy especially with the
growing need to find alternative methods for formulating
new type of safe and cost-effective antibiotics in
controlling the spread of resisted pathogens in food
processing environment (Jin et al., 2009; Rizwan et al.,
2010a). Some data suggest the selective toxicity of the
ZnO nanoparticles toward cancer cells (Shantikumar et
al., 2009). The anticancer effects of ZnO nanostructures
on human brain tumor U87 and cervical cancer Hela
were obtained and indicate promising activity that varies
with the changes in the structure and the size (Rizwan et
al., 2010b). Therefore, the present investigation was
aimed to determine the antibacterial activity of ZnO
nanoparticles toward E
coli as Gram-negative bacteria
and S. aureus as Gram-positive bacteria in laboratory
condition.

MATERIALS AND METHODS

Preparation of the materials and bacterial cultures

ZnO nanoparticles powder which were prepared by Amiri followed
by Nosaka method were used in this experiment (Nosaka et al.,
1998). The size of prepared ZnO nanoparticles was 3 nm.
Staphylococcus auerus PTCC 1431 and Escherichia coli PTCC
1399 were obtained from Persian Type Culture Collection. All these
strains were grown aerobically in nutrient broth for 24 h at 37°C
before using as target organisms. The density of bacterial isolates
was adjusted to an optimal density of 0.5 McFarland standards.

Antibacterial activity assay

In order to examine the antibacterial activity of the ZnO
nanoparticles on these microorganisms, ZnO nanoparticles were
suspended in sterile normal saline and constantly stirring until a
uniform colloidal suspension was formed to yield a powder
concentration of 1000 mg/ml. To assess toxicity range of ZnO
nanoparticles against E
coli and S. aureus, an appropriate volume
of test bacteria were inoculated in nutrient broth medium supple-
mented with serially diluted ZnO nanoparticles with two various
particle size and bulk suspensions, from 100 to 0.78 mg/ml. After
these experiments, the best range was proposed from 0.5 to 16
mg/ml of nanoparticle-free medium and bacteria-free medium were
used as control positive and control negative respectively. Colony
forming units (cfu) were quantified after an overnight incubation at
37°C. Mami-Karvani and Chehrazi 1369

Determination of zone of Inhibition

0.05 and 0.1 ml was added of various concentrations of two
different ZnO nanoparticle sizes and bulk ZnO in discs and wells,
respectively. After inoculation and cultivation of different target
bacteria on top of nutrient agar, discs and wells were placed in
selected area on different plates. The zone of inhibition (ZOI) was
measured after 24 h incubation. The antibacterial activity of two
different particle sizes and bulk ZnO were compared. To gain
different nanoparticle size‚ equal amount of synthesized ZnO
nanoparticles were dried at two different temperatures, 40 and
70°C. Increasing temperature resulted in bigger particle size.

Determination of minimum inhibitory concentration

MIC and MBC were measured using agar dilution tests
After
inoculation of target bacteria on nutrient agar with various
concentrations of ZnO nanoparticles, the growth rates of bacteria
were determined by counting colony forming unit (cfu) in each plate.
The plates which show no growth after 24 h incubation were
selected
0
1 ml of sterile distilled water was added to these plates
and transferred to fresh medium which had not any ZnO
nanoparticles. The lowest concentration from which the bacteria do
not grow when transferred to fresh medium is MBC and MIC is the
lowest concentration from which the colonies appeared on top of
fresh medium

Time dependent test

Time dependent tests were performed in nutrient broth
supplemented with different concentrations of ZnO nanoparticles
inoculated with the same amounts of test bacteria. Following
incubation at 37°C, 0.1 ml of different cultures was spread
separately on nutrient agar with respect of time. After 24 h, cfu was
quantified for each plate and compared with cfu in control plates. All
experiments were performed in triplicate and the averages were
obtained.

RESULTS AND DISCUSSION

The antibacterial activity of ZnO nanoparticles was tested
by the disc and well diffusion agar methods (Tables 1 and
2). The presence of an inhibition zone clearly indicated
the antibacterial effect of ZnO nanoparticles. As it was
also shown in the study of Rizwan et al. (2010c) it has
been seen in this study that by increasing the concen-
tration of ZnO nanoparticles in wells and discs, the
growth inhibition has also been increased. The size of
inhibition zone was different according to the type of
bacteria, the size and the concentrations of ZnO
nanoparticles.
Number of colony forming unit (cfu) of E. coli and S.
aureus after overnight incubation at the presence of
different concentrations of ZnO nanoparticles was shown
in Figure 1. The minimum concentration of ZnO
nanoparticles which inhibited the growth of bacteria was
3.1 mg/ml for E. coli and 1.5 mg/ml for S.aureus. This is
in agreement with previously published reports on the
antibacterial properties of ZnO nanoparticles which
showed that the minimum concentration at which the

1370 Afr. J. Microbiol. Res.

Table 1. Zone of inhibition (ZOI) for S. areus.

ZnO concentration in wells (mg/ml) ZOI (mm) ZnO concentration in discs(mg/ml) ZOI (mm)
10 29 5 22
5 27 2.5 19
2.5 25 1.25 16
1.25 21 0.625 14
0.625 17 0.312 12
0.312 15 0.156 10
0.156 14 0.078 9
0.078 *14 0.039 *9
0.039 0 0.0195 0
0.0195 0 0.00975 0
Control 0 Control 0

* Minimum concentrations of ZnO nanoparticles at which zone of inhibition started to appear.

Table 2. Zone of inhibition (ZOI) for E.coli.

ZnO concentration in wells(mg/ml) ZOI (mm) ZnO concentration in discs(mg/ml) ZOI (mm)
10 19 5 28
5 16 2.5 24
2.5 14 1.25 21
1.25 12 0.625 19
0.625 *10 0.312 *14
0.312 0 0.156 0
0.156 0 0.078 0
0.078 0 0.039 0
0.039 0 0.0195 0
control 0 Control 0

* Minimum concentrations of ZnO nanoparticles at which zone of inhibition started to appear.

growth of E. coli and S. aureus was inhibited was 3.4 and
1 mM, respectively (Reddy et al., 2007). The results of
MIC and MBC for E. coli and S. aureus were summarized
(Tables 3 and 4). Based on the results obtained from
MIC, MBC, disc and well agar diffusion methods, it can
be suggested that in comparison with Gram-positive
bacteria, the growth of gram-negative bacteria is inhibited
at higher concentrations of ZnO nanoparticles (Figure 2).
Reddy et al. (2007) have reported the same results,
emphasizing on the higher susceptibility of Gram-positive
bacteria in comparison with Gram-negative bacteria. In
the study done by Selahattin et al. (1998), it has been
proposed that the higher susceptibility of Gram-positive
bacteria could be related to differences in cell wall
structure, cell physiology, metabolism or degree of con-
tact. The results of time-dependant antibacterial activity
of ZnO nanoparticles showed that cfu of the tested
bacteria for each concentration decreased gradually
during 72 h, whereas colony formation of control solution
remained uncountable (Figures 3 and 4).
Significant differences was observed between antibacterial activity of bulk ZnO, yellow ZnO
nanoparticles dried at 70°C and white ZnO nanoparticles
dried at 40°C. The antibacterial efficacy increased with
decreasing particle size from bulk ZnO to white ZnO
nanoparticles (Figure 5). Particle concentration seems to
be more effective on the inhibition of bacterial growth
than particle size under the condition of this work (Figure
5) (Lingling et al., 2006). The enhanced bioactivity of
smaller particle probably is attributed to the higher sur-
face area to volume ratio (Nagarajan and Rajagopalan,
2008). According to the results, it can be concluded that
ZnO nanoparticles are effective antibacterial agents both
on Gram-positive and Gram-negative bacteria. The same
results were confirmed in the study of Zhongbing et al.
(2008) in which Gram-negative membrane and Gram-
positive membrane disorganization was approved by
transmission electron microscopy of bacteria ultrathin
sections. In order to use ZnO nanoparticles in in vivo
condition, further studies should be performed investi-
gating the toxic effect of ZnO nanoparticles on eukaryotic
cells. The study done by Alok and Vyom (2010)

Mami-Karvani and Chehrazi 1371

Figure 1. Number of colony forming units (cfu) of E. coli and S. aureus after overnight incubation at the presence of
different concentrations of ZnO nanoparticles.

Figure 2. Comparison of antibacterial activity of ZnO nanoparticles on
E.coli and S. Aureus

Table 3. Determination of MIC and MBC for E. coli.

Mode of effect Concentration (mg/ml)
Growth 0.125
Growth 0.25
Growth 0.5
Bacteriostatic 1(MIC)
Bacteriostatic 2
Bacteriostatic 4
Bacteriostatic 8
Bactericidal 16(MBC)

emphasized on the necessity of additional experiments
on the safety/toxicity properties of nanoparicles, due to Table 4. Determination of MIC and MBC for S. aureus.

Mode of effect Concentration (mg/ml)
Growth 0.125
Growth 0.25
Bacteriostatic 0.5(MIC)
Bacteriostatic 1
Bacteriostatic 2
Bacteriostatic 4
Bactericidal 8(MBC)
Bactericidal 16

the many experimental challenges encountered when
assessing the toxicity of them. The results of this study









1372 Afr. J. Microbiol. Res.

Figure 3. Number of colony forming units (cfu) of E. coli with respect of time.

Figure 4. Number of colony forming units (cfu) of S. aureus with respect of time.

Figure 5. Comparison of zone of inhibition for different particle size in E. coli.

also highlighted the need for understanding how ZnO
nanoparticles affect the bacterial cell and furthermore the
mechanism by which ZnO nanoparticles affect viable cell. It is proposed to work on the resistance mechanism of
resistant strains which were encountered in contact with
ZnO nanoparticles. It could be performed by studying the cfu/ml
16 mg/ml

8 mg/ml

4 mg/ml

2 mg/ml

1 mg/ml

0.5 mg/ml
control Zone of inhibition
(mm)
mg/ml

plasmid profile and identification of the resistant gene.

ACKNOWLEDGEMENT

We thank Dr. Gholamreza Amiri for preparing ZnO
nanoparticles for this study.

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