INTRODUCTION

The emergence of infectious diseases in general poses a serious threat to

public health worldwide (Azam et al., 2012). Generally, both Gram- positive and
Gram- negative bacterial strains are thought to present a major health problem.
Over the years, antibiotics have been used to control infections resulting from
both community and hospital environments (Ahmed et al., 2012). The need for
novel antibiotics comes from the relatively high incidence of bacterial infection
and the growing resistance of bacteria to conventional antibiotics. Bacteria, with
their large populations and fast reproduction time, are able to rapidly develop
mechanisms of antibiotic resistance when a subset of the bacterial population
survives antibiotic exposure (Singh et al., 2012). Antibiotic resistance may
develop via multiple mechanisms. Briefly, the primary mechanisms include
alteration or inactivation of the antibiotic by the bacteria, alteration of the target
site of the antibiotic, alteration of a metabolic pathway to avoid the disruptive
effect of the antibiotic, and reduced accumulation of the drug by minimizing its
entry or maximizing clearance from the cell (Reddy et al., 2007). To illustrate one
such mechanism, inactivation of the antibiotic, antibiotic resistance is due to the
ability of the bacteria to adapt production of beta-lactamase enzymes which
cleave the lactam ring, neutralizing beta-lactam antibiotics such as penicillin
(Reddy et al., 2007). Bacteria, either Gram- negative or Gram- positive may use
this mechanism alone, or in conjunction with other resistance mechanisms, to
dramatically reduce its susceptibility to the bactericidal effects of large classes of

traditional antibiotics.

Thus, new methods for reducing bacterial activity and

associated infections are badly needed (Seil, 2012). This growing resistance of
microorganisms to potent antibiotics has renewed a great interest towards
investigating bactericidal properties of nanoparticles and their nano- composites
as an alternative (Singh et al., 2012).
Nanotechnology, the use of materials with dimensions on the atomic or
molecular scale, has become increasingly applied to medical applications as an
approach to killing or reducing the activity of numerous microorganisms. While
some natural antibacterial materials, such as zinc and silver, possess greater
antibacterial properties as particle size is reduced into the nanometer regime (due
to the increased surface to volume ratio of a given mass of particles) [Yamamoto
2001, Dodd et al. 2006, Zhang et al. 2007], the physical structure of a
nanoparticle itself and the way in which it interacts with and penetrates into
bacteria appears to also provide unique bactericidal mechanisms (Seil, 2012).
Nanoparticles have one dimension that is 100 nanometers or less in size. The
formation of nanoparticles changes the properties of many conventional materials
(Reddy et al., 2007). For example, the larger the surface area of nanoparticles can
result in a greater degree of interaction with bacterial cell walls. Amongst
inorganic materials, metals and metal oxides have elicited a great deal of interest
for use as antimicrobial agents because of their durability, selectivity and
resistance to heat. One of the reasons why these inorganic materials (metal and
metal oxide) have attracted so much attention is their ability to withstand

intensive processing conditions (Fu et al., 2005; Makhluf et al., 2005). They have
selective toxicity to bacteria but have minimal effects on human cells (Reddy et
al., 2007).
Zinc oxide (ZnO) belongs to a group of metal oxides with photo-oxidising
and photocatalytic ability against chemical and biological species (Szab ., 2003).
It is an inorganic white powder and is insoluble in water. ZnO nanoparticles (NP)
have been shown to be useful antibacterial and antifungal agents when used as a
surface coating on materials and textiles (Abramov ., 2009). Zinc is an essential
element and ZnO nanoparticles are, interestingly, reported by other researches as
non-toxic to human cell (Sirelkhatim, 2012). Thus, this study was considered
inorder to determine the Zinc oxide nanoparticles synthesized from the
Department of Physics of MSU-Iligan Institute of Technology.

Objectives of the Study:

1. To determine the minimum concentration of Zinc oxide nanoparticle
suspension that inhibits the growth of Staphylococcus aureus,
Salmonella typhimurium, Escherichia coli, and Bacillus subtilis using
the Tube Dilution Assay.
2. To establish at which concentrations (0.025, 0.03, 0.05 and 0.1) mg/ml
of ZnO NP that will show the most effective antibacterial activity
using the modified Kirby-Bauer disk diffusion method.

3. To determine the test organism that is most susceptible to the

antibacterial effect of Zinc oxide NP.

Significance of the Study:

This study will provide significant information to the medical,
pharmacological, academe, and industrial world regarding the efficacy of Zinc
oxide nanoparticles as antimicrobial agents. Nonetheless, this study is essential as
it provides low- cost antibacterial material especially in the food industry and in
the water supply system since this ZnO NP could be used in the metal coatings of
pipes and containers.

Scope and Limitations of the Study

This study was focused on determining the minimum concentration of
Zinc oxide NP suspension that inhibits the growth of S. aureus, S. typhimurium,
E. coli, and B. subtilis using the Kirby- Bauer modified disk diffusion method.
By using this test, the concentration that had the most effective antimicrobial
property was also identified. Also, the test organism most susceptible to the test
material was also determined. However, the ZnO nanoparticle used in this study
was limited only to rod shaped zinc oxide nanoparticles. The test organisms used
were limited only to two Gram- positive (S. aureus and B. subtilis) and two Gramnegative bacteria (E.coli and S. typhimurium).

Moreover, this study was

conducted in the General Laboratory-2 (GL-2) of the College of Science and

Mathematics, MSU-IIT and the materials used were obtained within the city of
Iligan except for the test organisms that were purchased from the Philippine
National Collection of Microorganisms, National Institute of Molecular Biology
and Biotechnology, University of the Philippines Los Baos.

MATERIALS AND METHODS

A.

Place and Duration of the Study

This study was conducted in the General Laboratory 2, Department of

Biological Sciences, College of Science and Mathematics, MSU- Iligan Institute

of Technology, Iligan City from January 2014 until March 2014.

B.

Determination of Minimum Inhibitory Concentration

Tube Dilution Assay was used to determine the minimum inhibitory

concentration of the ZnO nanoparticles (Gunalan, 2012). Twenty-four sterile test

tubes were prepared. Six tubes for each of the four test organism that were labeled
1 through 6. Meanwhile, a ZnO nanoparticles solution was prepared by dissolving
1000g of ZnO nanoparticles in 10ml of ddH 2O (100g/ml). Two milliliters of
the ZnO solution was then added to Tube No. 1 of all four sets while 1mL of
sterile Nutrient Broth to all other tubes. Done uniformly to all four sets of tubes,
1mL was transferred from Tube 1 to Tube 2. Contents of Tube 2 were mixed and
using a different pipette tip, 1mL was transferred to Tube 3. Dilutions were
continued in this manner to Tube 5, being certain to change pipette tips between
tubes to prevent carryover of the ZnO solution on the external surface of the
pipette tip. One milliliter was removed from Tube 6 and was discarded. Tube 6,

which serves as the control, received no ZnO solution. The ZnO concentration of
in each of the tubes was then half the concentration of the preceding tube100g/mL, 50g/mL, 25g/mL, 12.5g/mL, and 6.25g/mL. Several colonies of
the test organisms were suspended to an appropriate turbidity in 5mL of MuellerHinton broth to give a slightly turbid suspension. This suspension was then
diluted by aseptically pipetting 0.2mL of the suspension into 40mL of MuellerHinton broth. One milliliter of the diluted culture suspension was then added to
each of the thirty-six tubes. All tubes were then incubated overnight for
examination for visible signs of microbial growth.

C.

Collection of Test Organisms

Pure cultures of Staphylococcus aureus, Bacillus subtilis, Escherichia coli,

and Salmonella typhimurium were purchased from the Philippine National

Collection of Microorganisms, National Institute of Molecular Biology and
Biotechnology, University of the Philippines Los Baos. The test organisms
arrived three weeks after purchase. To ensure that the organisms were pure and
contaminant-free, the PNCM placed the culture in a screw- cap tube that was
securely wrapped using a bubble wrap.

D.

Preparation of Stock Suspension of Zinc Oxide nanostructures

ZnO nanostructures were obtained from the Physics Department of the

Mindanao State University- Iligan Institute of Technology. The size and shape of
the nanoparticle were identified using the Scanning Electron Microscope from the
Material Science Laboratory of the Physics Department. The SEM results took
three weeks to arrive. Meanwhile, a stock suspension was prepared by preparing
four 20mL test tubes and filling them with 10mL of double distilled water
(ddH2O) (Xie, 2011). Also, the ZnO nanoparticle was also weighed using the
digital laboratory weighing scale into the following measurements: 0.25mg,
0.3mg, 0.5mg, and 0.1mg. The weighed nanoparticle was then suspended into
each of the dispensed double distilled water to yield the concentrations: 0.025,
0.03, 0.05, and 0.10 mg/ml (Xie, 2011).

The suspensions were then mixed

thoroughly using vortex, homogenous mixtures of the suspensions containing the

specific ZnO concentrations were obtained.

E.

Antimicrobial Activity Testing

To identify the antimicrobial property of ZnO nanoparticle, the

antibacterial assay was carried out as described by Azam (2012) with little
modifications.

E.1. Preparation of Test Organisms

Pure cultures of the test organisms from PNCM were subcultured in

Nutrient Broth for bacterial culture revival by filling four 20mL test tubes with
cotton plugs with 10mL of cooked Nutrient Broth. Using a sterile inoculation
loop, a pure colony of each of the test organisms (S. aureus, S. typhimurium, E.
coli, and B. subtilis) was picked from the pure culture and then transferred to each
of the prepared Nutrient Broth with name labels.

E.2. Antimicrobial Assay

Antimicrobial activities of the synthesized ZnO nanoparticles were
performed against both Gram-negative (E. coli and S. typhimurium) and Grampositive (S.aureus and B. subtilis) bacteria. The antimicrobial activity was done
by modified Kirby-Bauer Disk Diffusion Method. For the antimicrobial assay, a
lawn of culture was prepared by spreading 100 L of the fresh cultures of the test
organisms from D.1. on Mueller Hinton Agar (MHA) plates with the help of a
sterile glass-rod spreader (Azam, 2012). The MHA plates were then left to stand
for ten minutes to allow the culture to get absorbed by the agar. After ten minutes,
using a sterile glass tubing, six 8mm wells were punched onto the MHA plates
for the nanostructure antimicrobial activity testing. It was made sure that the
wells were of uniform distances to minimize error of inhibition zone comparison.
One drop of molten agar was then used to seal the wells to avoid leakage of
nanoparticles from the bottom of the wells. After all the wells created were made

10

secure, using a micropipette, 100 L of the nanoparticle suspension was poured

onto each of the four wells on all the plates (one well for each of the four
concentrations of the nanoparticle suspension). The two remaining wells were
used for the negative (distilled water) and positive (Chloramphenicol for Gramnegative; Streptomycin for Gram-positive) controls.

E.3. Observation of Inhibition Zones

After overnight (16 hours) incubation, the zones of inhibition were then
measured using a Carbon Fiber Dial Caliper. Inhibition zones created around
each of the six wells were measured and recorded for comparison.

F.

Statistical Analysis
Data recorded were analyzed using SPSS, ver. 10.0. The analysis of

variance (ANOVA) was used to assess the significant differences between the
control and each of the treatment. If there was a significant difference (P<0.05),
the experimental data were analyzed further using Duncans Multiple Range Test
(Azam, 2012).

RESULTS AND DISCUSSION

11

In order to determine the minimum inhibitory concentration (MIC) of ZnO

nanoparticle used in this study, a tube dilution assay was done (Gunalan, et al,
2009). When you mix the bacteria growing in a liquid medium, the culture
appears turbid (Martinko, 2002). This is because a bacterial culture acts as a
colloidal suspension that blocks and reflects light passing through the culture. The
concentrations identified were 100g/mL, 50g/mL, 25g/mL, 12.5g/mL, and
6.25g/mL. MIC is determined as the lowest nanoparticle concentration that did
not permit any visible growth of microorganisms during 24 hours of incubation on

Table 1. Minimum inhibitory concentration of ZnO nanoparticle against Gramnegative and Gram-positive bacteria.
ZnO

Gram-negative bacteria

Gram-positive bacteria

concentration

S.typhimurium

E.coli

B.subtilis

S.aureus

6.25g/mL

Turbid

Turbid

Turbid

Turbid

12.5g/mL

Turbid

Turbid

Clear**

Clear**

25g/mL

Turbid

Turbid

Clear

Clear

50g/mL

Clear **

Clear**

Clear

Clear

100g/mL

Clear

Clear

Clear

Clear

Control

Turbid

Turbid

Turbid

Turbid

** designates the Minimum Inhibitory Concentration.

the basis of turbidity (Sirelkhatim, 2012). In other words, it is the concentration

which impedes and absolutely prevents bacterial growth. It was found to be
>12.5g/mL and >50g/mL for the Gram-positive and Gram-negative test
organisms, respectively, as shown in Table 1. These results were consistent with

12

those obtained by Xie et al. (2011) which suggested MIC of 12g/mL and
48g/mL for the Gram-positive and Gram-negative microbes.
However, to assess the antibacterial property of each of the ZnO
nanoparticle concentrations, disk-diffusion method was also used. The ZnO
concentrations tested were identified to be 0.025 mg/ml, 0.03 mg/ml, 0.05 mg/ml
and 1.0 mg/ml as seen in Figure 1.
Inhibitory activity was determined against Gram-negative bacteria (Figure
1). Results showed that the greatest inhibitory effect was observed on the
nanoparticle suspension with concentration of 0.1 mg/ml against S. typhimurium
and E. coli with a zone of inhibition of 12.8 mm and 13.7 mm respectively. This
said concentration showed a significant difference at p<0.05 when compared to
the control. It was also observed that at 0.025 nanoparticle suspension
concentration, the results showed no significant difference with the control. Thus,
it was demonstrated that this concentration is still efficient as it is at par with the
control which is the standard antibiotic.

13

d d
a a
a a

b b

Figure 1. Inhibitory activity of ZnO nanoparticle against Gram-negative

bacteria. Different letters mean significant difference at p<0.05. Positive control
used is the antibiotic, Chloramphenicol.

Inhibitory effect against Gram- positive bacteria was also determined

(Figure 2). The results demonstrated that the nanoparticle suspension with
concentration of 0.1mg/ml exhibited the greatest inhibitory effect against B.
subtilis and S. aureus with zones of inhibition of 21mm and 20.8mm, respectively.
This concentration presented a significant difference in comparison with the
control and the other concentrations at p<0.05. However, nanoparticle suspension
concentration of 0.025mg/ml showed no significant difference with the control.
This demonstrated that at this concentration, ZnO nanoparticle is as efficient with
the standard antibiotic.

14

As it was also shown in the study of Rizwan et al. (2010), it has been seen
in this study that as the nanoparticle suspension was increased, the zone of
inhibition also increased. This is due to the damaging effect of ZnO nanoparticles
with the bacterial cells and increased production of active hydrogen such as H 2O2
that lead to cell death. Vani et al (2008) presented that ZnO nanoparticles, after its
adherence to the surface of the cell membrane, results in disturbance in its
respiration as it interact with enzymes of the respiration chains of bacteria. These

Figure 2. Inhibitory Effect of ZnO Nanoparticle against Gram-positive bacteria.

Different letters means significant difference at p<0.05. Positive control used is
the antibiotic, Streptomycin.

15

results were also in accordance with Banoee et al (2010), who reported that
inhibition- zone increased significantly with increasing ZnO nanoparticle
loadings. Thus, the present results indicated that the growth of the test organisms
was much affected by the ZnO nanoparticles.
Nanoparticle size and concentration play important roles in the
antibacterial activity. SEM images of the ZnO nanoparticle used, as shown in
Appendix D, were obtained to determine the size and shape of the nanoparticle.
Sirelkhatim (2013) presented that ZnO nanoparticles of smaller sizes can easily
penetrate into bacterial membranes due to their large interfacial area, thus
enhancing their antibacterial efficiency. A large number of studies investigated on
the considerable impact of particle size on the antibacterial activity, and the
researchers found that controlling ZnO nanoparticle size was crucial to achieve
best antibacterial response, and ZnO nanoparticles with smaller sizes (higher
specific surface areas) showed highest antibacterial activity (Sawai et al., 1996).
The effect of concentration and size was successfully analyzed by a work
carried out by Padmavathy and Vijayaraghavan (2008) who described the
antibacterial activity of ZnO nanoparticles to be dependent mainly on the surface
area of ZnO.

Furthermore, a correspondence between nanoparticle size and

concentration appears to be required for the bioactivity of ZnO nanoparticles. It

was also concluded by Yamamoto, through his examination of the influence of
ZnO nanoparticles size on the antibacterial activity against S. aureus and E.coli,
that decrease in particle size will increase the antibacterial activity.

16

It should also be noticed that Gram- negative strains of E. coli and S.

typhimurium had inhibition- zone sizes that were lower than Gram- positive
bacterial strains of B. subtilis and S. aureus as demonstrated in Figure 3. In all
data presented, it is clear that the growth inhibition for the Gram-negative bacteria
occurred at higher ZnO concentrations.

Figure 3. Inhibition-zone sizes of Gram- negative and Gram-positive bacteria.

This observation could be indicative of higher Gram- negative strain

tolerance against Zinc oxide nanoparticles over Gram- positive bacterial strains.
The finding is in agreement with Premanathan et al (2011), who reported that the
ZnO nanoparticle effect is more pronounced against Gram- positive bacterial
strains than Gram- negative bacterial strains. In the study done by Selahattin et al.
(1998), it has been proposed that the higher susceptibility of Gram- positive

17

bacteria could be related to differences in cell wall structure, cell physiology,

metabolism or degree of contact.

CONCLUSION AND RECOMMENDATION

Based on the results, Zinc oxide nanoparticles showed a very good degree
of antibacterial activity against both Gram- positive and Gram- negative bacteria.
It was also clearly demonstrated that the least ZnO nanoparticle concentrations
that inhibited bacterial growth were 12.5 ug/ml for Gram-negative and 50 ug/ml

18

for Gram-positive bacteria. The best concentration that had the most effective
antibacterial property, on the other hand, was 0.1 mg/ml. The results also
presented that higher antibacterial activity was observed against Gram- positive
bacteria than the Gram- negative bacteria.
This study recommends the examination of the mechanisms of the
inhibitory activity of the ZnO nanoparticles at the molecular level. It is also
recommended that inhibitory effect of ZnO nanoparticles against other Gramnegative and Gram- positive organisms, fungi, and eukaryotes like parasites be
studied, as well. The use of spectrophotometer in the identification and analysis
of the minimum inhibitory concentration based on turbidity is also encouraged as
it indirectly measures all bacteria (cell biomass), dead or alive. Also, that to avoid
possibility of misinterpretations on turbidity, it is recommended to subculture the
serial dilution in agar plates.

LITERATURE CITED

A.L. Barry, W.A. Craig, H. Nadler, L.B. Reller, C.C. Sanders, J.M. Swenson.
1999. Methods for Determining Bactericidal Activity of Antimicrobial
Agents; Approved Guideline, vol. 19, 18th edn. (National Committee for
Clinical Laboratory Standards, CLSI, Wayne)

19

Arshad M, Azam A, Ahmed AS, Mollah S, Naqvi AH. 2012. Effect of Co

substitution on the structural and optical properties of ZnO nanoparticles
synthesized by solgel route. J Alloys Compd. 509:83788381.
Baek Y, An Y. 2011. Microbial toxicity of metal oxide nanoparticles (CuO, NiO,
ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus
aureus. Sci Total Environ. 409:16031608.
Bauer AW, Kirby WMM, Sherris JC, Truck M. 1966. Antibiotic susceptibility
testing by standardized single disk method. Am J Clin Pathol. 45: 493
496.
Berry CC, Curtis ASG. 2003. Functionalisation of magnetic nanoparticles for
applications in biomedicine. J Phys D Appl Phys. 36: R198R206.
Brayner R. 2008. The toxicological impact of nanoparticles. Nano Today. 3:48
55.
Chastellain M, Petri A, Hofmann H. 2004. Particle size investigations of a
multistep synthesis of PVA coated superparamagnetic nanoparticles. J
Colloid Interface Sci. 278(2):353360.
Daniel L, Bo L, Kenneth HB. 2003. Absorption of zinc from wheat products
fortified with iron and either zinc sulfate or zinc oxide. Am. J. Clin. Nutr.,
78:279-283.
Gajjar P, Pettee B, Britt DW, Huang W, Johnson WP, Anderson AJ. 2009.
Antimicrobial activities of commercial nanoparticles against an
environmental soil microbe, Pseudomonas putida KT2440. J Biol Eng.
3:9.
Grinholc M, Szramka B, Kurlenda J, Graczyk A, Bielawski KP. 2008.
Bactericidal effect of photodynamic inactivation against methicillinresistant and methicillin-susceptible Staphylococcus aureus is straindependent. J Photochem Photobiol A Chem. 90(1): 5763.
Hawkey PM. 2008. The growing burden of antimicrobial resistance. J Antimicrob
Chemother. 62 Suppl 1:i1i9.
Heinlaan M, Ivask A, Blinova I, Dubourguier HC, Kahru A. 2008. Toxicity of
nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and

20

crustaceans Daphnia magna

Chemosphere. 71:13081316.

and

Thamnocephalus

platyurus.

Jones N, Ray B, Ranjit KT, Manna AC. 2008. Antibacterial activity of ZnO
nanoparticles suspensions on a broad spectrum of microorganisms. FEMS
Microbiol Lett. 279:7176.
J. Sawai, E. Kawada, F. Kanou, H. Igarashi, A. Hashimoto, T. Kokugan, M.
Shimizu. 1996. Detection of active oxygen generated from ceramic
powders having antibacterial activity. J. Chem. Eng. Jpn. 29(4), 627633
doi:10.1252/jcej.29.627
J. Sawai. 2003. Quantitative evaluation of antibacterial activities of metallic oxide
powders (ZnO, MgO and CaO) by conductimetric assay. J. Microbiol.
Methods 54(2), 177182 doi:10. 1016/S0167-7012(03)00037-X
Keenan CR, Sedlak DL. 2008. Factors affecting the yield of oxidants from the
reaction of nanoparticulate zero-valent iron and oxygen. Environ Technol.
42(4):12621267.
K.M. Reddy, K. Feris, J. Bell, D.G. Wingett, C. Hanley, A. Punnoose. 2007.
Selective toxicity of zinc oxide nanoparticles to prokaryotic and
eukaryotic
systems.
Appl.
Phys.
Lett.
90(21),
213902.
doi:10.1063/1.2742324
Kohler N, Sun C, Wang J, Zhang M. 2005. Methotrexate-modified
superparamagnetic nanoparticles and their intracellular uptake into human
cancer cells. Langmuir. 21(19):88588864.
Komolafe OO. 2003. Antibiotic resistance in bacteria an emerging public health
problem. Malawi Med J. 15:6367.
Lee C, Kim JY, Lee WI, Nelson KL, Yoon J, Sedlak DL. 2008. Bactericidal effect
of zero-valent iron nanoparticles on Escherichia coli. Environ Technol.
42(13):49274933
Martinko J, Bender K, Buckley D. 2002. Brock Biology of Microorganisms 14 th
Edition
Padmavathy N, Vijayaraghavan R. 2008. Enhanced bioactivity of ZnO
nanoparticles - an antimicrobial study.Sci Technol Adv Mater. 9:17.

21

Premanathan M, Karthikeyan K, Jeyasubramanian K, Manivannan G. 2011.

Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and
cancer cells by apoptosis through lipid peroxidation. Nanomedicine.
7:184192.
Raghupati KR, Koodali RT, Manna AC. 2011. Size-dependent bacterial growth
inhibition and mechanism of antibacterial activity of zinc oxide
nanoparticles. Langmuir. 27:40204028.
Reddy KM, Kevin F, Jason B, Cory H, Alex P. 2007. Selective toxicity of zinc
oxide nanoparticles to prokaryotic and eukaryotic systems. Appl. Phys.
Lett., 90:1-8.
Rosi NL, Mirkin CA. 2005. Nanostructures in biodiagnostics. Chem Rev.
105:15471562.
Roselli M, Finamore A, Garaguso I, Britti MS, Mengheri E. 2003. Zinc oxide
protects cultured enterocytes from the damage induced by Eschrichia Coli.
J. Nutr., 133:4077-4082
Salah N, Habib SS, Khan ZH, et al. 2011. High-energy ball milling technique for
ZnO nanoparticles as antibacterial material. Int J Nanomedicine. 6:863
869.
Sawai J, Shinobu S, Igarashi H, Atsushi H, Takao K, Masaru S, Hiromitsu K.
1998. Hydrogen Peroxide as an Antibacterial Factor in Zinc Oxide Powder
Slurry. J. Ferment. Bioeng., 86:521-522
Soderberg TA, Sunzel B, Holm S, Elmros T, Hallmans G, Sjoberg S. 1990.
Antibacterial effect of zinc oxide in vitro. Scand. J. Plast. Reconstr. Surg.
Hand Surg., 24:193-197.
Touati D. 2000. Iron and oxidative stress in bacteria. Arch Biochem Biophys.
373(1):16
Tran N, Mir A, Mallik D, Sinha A, Nayar S, Webster TJ. 2010. Bactericidal
effects of iron oxide nanoparticles on Staphylococcus aureus. Int J
Nanomedicine. 5:277283.
Wang Z, Lee Y, Wu B, et al. 2010. Anti-microbial activities of aerosolized
transition metal oxide nanoparticles. Chemosphere. 80: 525529.

22

Wikler MA. 2000. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically: Approved Standard. 5th ed. Wayne, PA:
National Committee for Clinical Laboratory Standards (NCCLS); M7
M5.
Wu W, Xiao XH, Peng TC, Jiang CZ. 2010. Controllable synthesis and optical
properties of connected zinc oxide nanoparticles. Chem-Asian. J., 5:315321
Xie Y, He Y, Irvin PL, Jin T, Shi X. 2011. Antibacterial activity and mechanism
of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl
Environ Microbiol. 77:23252331.
Xu M, Fujita D, Kajiwara S, et al. 2010. Contribution of physicochemical
characteristics of nano-oxides to cytotoxicity. Biomaterials. 31: 8022
8031.
Yamamoto O. 2001. Influence of particle size on the antibacterial activity of zinc
oxide. Inter J Inorg Mater. 3(7):643646.
Zhang L, Jiang Y, Ding Y, Povey M, York D. 2007. Investigation into the
antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO
nanofluids). J Nanopart Res. 9(3):479489.
Z. Xu, J.-Y. Hwang, B. Li, X. Huang, H. Wang. 2008. The characterization of
various ZnO nanostructures using eld-emission SEM. JOM 60(4), 2932
doi:10.1007/s11837-008-00449

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APPENDICES

APPENDIX A

Appendix A.1. ANOVA results of the effect of zinc oxide nanoparticles

concentrations on the growth of Salmonella typhimurium.
ANOVA Table for Salmonella growth inhibition

Sum of
Squares

df

Mean
Square

Zinc oxide concentration

155.696

38.924

Error

377.522

40

9.438

Total

533.218

44

p-value

4.124

.007

24

Appendix A.2. Duncans Multiple Range Test results of Salmonella growth

inhibitions at different Zinc oxide nanoparticle concentration.
Multiple Comparisons
Control (I)

(J)
ZincConc

Mean Difference
(I-J)

Std. Error

p-value

0.025

-2.788888889

1.4482

0.45789

0.03

-0.144444444

1.4482

0.99999

0.05

-2.444444444

1.4482

0.58844

0.1

-5.011111111

1.4482

0.02980*

Streptomycin

Remark
Not
Significa
nt
Not
Significa
nt
Not
Significa
nt
Significa
nt

*The mean difference is significant at the 0.05 level.

APPENDIX B

Appendix B.1. ANOVA results of the effect of zinc oxide nanoparticles

concentrations on the growth of Escherichia coli.

25

ANOVA Table for E. coli growth inhibition

Sum of

Mean

df

Squares

Sig.

4.564

.004

Square

Zinc oxide concentration

112.006

28.001

Error

245.402

40

6.135

Total

357.408

44

Appendix B.2. Duncans Multiple Range Test results of E. coli growth inhibitions
at different Zinc oxide nanoparticle concentration.

Multiple Comparisons
(I) Control

Streptomyci
n

(J)
ZincOnc

Mean Difference
(I-J)

Std. Error

Sig.

0.025

-0.72222222

1.167624

0.983274

0.03

-2.51111111

1.167624

0.344471

0.05

-3.06666667

1.167624

0.163602

0.1

-4.34444444

1.167624

0.016098

*The mean difference is significant at the 0.05 level.

APPENDIX C

Remark
Not
Significan
t
Not
Significan
t
Not
Significan
t
Significan
t

26

Appendix C.1. ANOVA results of the effect of zinc oxide nanoparticles

concentrations on the growth of Bacillus subtilis.
ANOVA Table for B. subtilis growth inhibition

Sum of

Mean

Squares

df

Square

Sig.

Zinc oxide concentration

669.248

167.312

45.559

.000

Error

146.896

40

3.672

Total

816.143

44

Appendix C.2. Duncans Multiple Range Test results of B. subtilis growth

inhibitions at different Zinc oxide nanoparticle concentration.

Multiple Comparisons
(I) Control

Streptomycin

(J)
ZincOnc

Mean
Difference (I-J)

Std. Error

Sig.

Remark

0.025

-0.68889

0.903375

0.963996

Not
Significant

0.03

-3.94444

0.00307

Signific
ant

Significant

0.05

-3.44444

0.903375

0.0128

0.1

-10.8778

0.903375

*The mean difference is significant at the 0.05 level.

APPENDIX D

Significant
Significant

27

Appendix D.1. Scanning Electron Microscopy image of the Zinc oxide

nanoparticle at 10m scale.

Appendix D.2. Scanning Electron Microscopy image of the Zinc oxide

nanoparticle at 5m scale.

28

Appendix D.3. Scanning Electron Microscopy image of the Zinc oxide

nanoparticle at 1m scale.

29