Journal of Biomaterials and Nanobiotechnology, 2012, 3, 480-485
http://dx.doi.org/10.4236/jbnb.2012.34049 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)
Comparative Study on Bactericidal Effect of Silver
Nanoparticles, Synthesized Using Green Technology, in
Combination with Antibiotics on Salmonella typhi
Shweta Rajawat, Mohammad Shums Qureshi
Department of Physics, Maulana Azad National Institute of Technology, Bhopal, India.
Received March 12th, 2012; revised June 25th, 2012; accepted July 24th, 2012
ABSTRACT
In this work bactericidal study of silver nanoparticles was taken up in combination with two standard antibiotics, am-
picillin and gentamycin, for Salmonella typhi. The antibacterial activities of antibiotics were increased in the presence
of AgNPs against test strains. The higher enhancing effect was observed for ampicillin in comparison to gentamicin
against test strains. Silver nanoparticles were synthesized elctrolytically using silver wire of 99% purity as anode and
carbon rod wrapped with LDPE as cathode. Silver nitrate [of Merck] of 0.01N is used as an electrolyte. Here tea extract
is added as capping and mild reducing agent. The polyphenols theaflavins and thearubigins, present in tea perform the
role of stabilizing or capping agents due to their bulky and steric nature. A brown coloured colloidal solution of silver
nanoparticles is obtained. The as-synthesized silver nanoparticles were characterized using XRD, TEM and UV-Vis
spectroscopy.
Keywords: Silver Nanoparticles; Salmonella typhi; Tea Extract; Ampicillin; Gentamicin
1. Introduction
Historically, silver has been extensively used for both
hygienic and healing purposes (Cheng and Schluesener,
2008). With time, the use of silver has reduced as an
anti-infection agent due to the advent of antibiotics and
other disinfectants and the poorly understood mecha-
nisms of their toxic effects. However, resistance of bac-
teria to bactericides and antibiotics has increased in re-
cent years. Some antimicrobial agents are extremely irri-
tant and toxic. Hence there is a need to find ways to for-
mulate safe and cost-effective Biocidal materials. Previ-
ous studies show that antimicrobial formulations of silver
in the form of nanoparticles could be used as effective
bactericidal materials. Highly reactive metal oxide nano-
particles exhibit excellent biocidal action against Gram-
positive and Gram-negative bacteria [1]. Thus, the syn-
theses, characterization, functionalization of nanosized
particles open the possibility of formulation of a new
generation of bactericidal materials.
Researchers have found that nanosilver and antibiotics
used together had an extremely strong effect against
gram positive and gram negative bacteria. Recently, due
to the limitations of antibiotics, the synergetic effect of
silver nanoparticles with antibiotics has been studied
combining silver nanoparticles with different antibiotics
against gram positive and gram negative bacteria.
A variety of preparation routes have been reported for
the synthesis of metallic nanoparticles [2,3] notable ex-
amples include, reverse micelles process [4,5], salt re-
duction [6], microwave dielectric heating reduction [7],
ultrasonic irradiation [8], radiolysis [9], Solvothermal
synthesis [10], electrochemical synthesis [11,12], etc.
Chemical methods for metal nanoparticle fabrication
usually involve toxic chemicals, which are usually ex-
pensive and potentially dangerous for the environment
[13]. Green synthesis of nanoparticles is an easy, effi-
cient and eco-friendly approach. Among several synthe-
sizing methods, biosynthetic methods employing either
biological microorganisms or plant extracts have emerg-
ed as a simple and viable alternative to chemical syn-
thetic procedures and physical methods. The synthesis of
metal nanoparticles using biological systems is an ex-
panding research area due to the potential ap plications in
nano medicines. Plant extracts play an important role in
remediation of toxic metals through reduction of the
metal ions. Silver nanoparticle are synthesized from
various parts of the herbal plants like bark of Cinnamon
(Sathishkumar et al., 2009), Neem leaves (Tripathi et al.,
2009), Citrus limon (Prathna et al., 2011), Tannic acid
(Sivaraman et al., 2009) and various plant leaves (Song

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,
in Combination with Antibiotics on Salmonella typhi 481
and Kim, 2008).
We describe a synthesis approach which is simple and
“green” for the synthesis of metallic nanostructures of
noble metal i.e. silver (Ag). The bactericidal effect of
as-synthesized silver nanoparticles is then studied for
combination with gentamycin and ampicillin for Salmo-
nella typhi. Salmonella typhi is an obligate parasite that
has no known natural r eservoir outside of humans. It is a
multi-organ pathogen that inhabits the lymphatic tissues
of the small intestine, liver, spleen, and bloodstream of
infected humans. Infection of S. typhi leads to the de-
velopment of typhoid, or enteric fever. Gentamicin is
active against a wide range of human bacterial infections,
mostly Gram-negative bacteria including Pseudomonas,
Proteus, Serratia and the Gram-positive Staphylococcus.
Ampicillin is a beta-lactam antibiotic that has been used
extensively to treat bacterial infections since 1961. It is
relatively non-toxic. The toxicity of silver nanoparticles
synthesized using green technology is comparatively
reduced.
2. Materials and Method
The experimental setup consists of a beaker filled with
40 ml of electrolyte silver nitrate (0.02 N) of MERK. The
silver nitrate was further diluted with triple deionised
distilled water to obtain a solution of 0.01 N (mol/l)
strength. Two electrodes: silver wire (99% pure) as an-
ode and carbon rod wrapped by LDPE (Low Density
Poly Ethylene) material as cathode was used. LDPE ma-
terial is used to collect silver nanoparticles produced in
the synthesis process so that they can be easily extracted.
The distance between the two electrodes is 1 cm. The
diameter of the silver wire is 1.04 mm and the diameter
of the carbon rod used is 4 mm. The length of the carbon
rod as well as silver wire is 4.5 cm. 2 ml of tea extract is
added to the electrolyte as a capping agent. The whole
assembly is placed on magnetic stirrer which keeps the
solution in the beaker stirring continuously. The process
is carried at room temperature (30˚C) for 2 hours con-
tinuously. A Daniel cell of 1.1 volt and 2 ohm internal
resistance is used as current source. A potentiometer pot
along with a rheostat is used in the circuit to increase the
resistance and obtain current of different values in mam-
peres. Copper wires are used to connect the components
of the circuit. All the parameters were same for the sam-
ples synthesized, except the current through the circuit.
Figure 1 shows the set up. The beaker in the set-up is
covered with silver foil with holes for the electrodes.
3. Results and Discussions
The as-synthesised silver nanoparticles were obtained in
the form of colloidal solution of light brown colour as
shown in Figure 2. The as-synthesized silver nanoparti-
cles were characterized using XRD. Figure 3 shows the
graph obtained. The XRD results show Face-Centred-
Structure of pure silver nanoparticles oriented in planes
{111}, {200}, {220 }, and {311}. The peaks obtained for
as-synthesized silver nanoparticles are well in accor-
dance with JCPDS file No. 04-0783. The intensive dif-
fraction peak from the {111} lattice plane of face-centred
cubic silver indicates that the particles are made of pure
silver and that their basal plane, i.e., the top crystal plane
Figure 1. Experimental setup.
Figure 2. Synthesized colloidal solution of silver nanoparti-
cles.

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,
in Combination with Antibiotics on Salmonella typhi
482
Figure 3. XRD graph of as-synthesized silver nanoparticles.
is the {111} plane. This 2θ value reflection angle con-
firms the presence of silver nanoparticles.
The morphology and the crystal structure of synthe-
sized silver nanoparticles were examined using HR-TEM.
HR-TEM unit used had a 120 kV class high-resolution
transmission electron microscope JEM-1400, Jeol, Japan.
The sample was placed on the carbon coated copper grid,
making a thin film of sample on the grid and extra sam-
ple was removed using the cone of a blotting paper and
kept in grid box sequentially. The TEM images show that
the particles are well dispersed and spherical in shape.
The particle size is between 2 nm to 23 nm. The average
size of the particle is 10 nm. The as-synthesized silver
nanoparticles are polydispersed nanoparticles. Such va-
riation in shape and size of nanoparticles synthesized by
biological systems is common [14]. The spherical and
oval shape of the particle, as visible in Figure 4, is due to
the fact that when a particle is formed, in its initial state,
it tries to acquire a shape that corresponds to minimum
potential energy. The spherical and oval shapes corre-
spond to the state of minimum potential energy. Other
factors that play role in determining the shape of silver
nanoparticles are the fast rate of reaction and low con-
centration of silver nitrate solution as slower rate of reac-
tion [15] and higher concentration of silver nitrate [16]
leads to anisotropic silver nanoparticles.
The UV-vis spectroscopy was carried by Uv-vis spec-
trometer of Systronics. It is a dual beam spectropho-
tometer. The base solution was triple de-ionised distilled
water to sample was added in small concentration. The
UV-visible spectrum shows the formation of silver
nanoparticles as the peak maxima 522 n m which is char-
acteristic to silver nanoparticles. The specific character-
istic peak, as shown in the Figure 5, for silver nanoparti-
cles is due to the surface Plasmon resonance. The nano-
particles which are smaller than the wavelength of light
can produce a coherent resonance waves at a particular
Figure 4. TEM image of silver nanoparticles.
Figure 5. UV-Visible graph of silver nanoparticles.
absorbance wavelength which is in the visible range for
silver nanoparticles.
Silver is known for its antimicrobial properties and h as
been used for years in the medical field for antimicrobial
applications and even has shown to prevent HIV binding
to host cells [17,18-21]. Additionally, silver has been
used in water and air filtration to eliminate microorga-
nisms [22-24].The antibacterial activity of nanoparticle
along with antibiotics ampicillin and gentamicin was
tested against S. typhi. Figure 6 shows the zone of inhi-
bition against S. typhi.The antibacterial activities of
antibiotics increase in the presence of silver nanoparticles
against gram positive and gram negative bacteria. The
size of metallic nanoparticles ensures that a significantly
large surface area of the particles is in contact with the
bacterial cells. Such a large contact surface is expected to
enhance the extent of bacterial elimination [25]. The
mechanism of the bactericidal effect of AgNPs is that
they may attach to the surface of the cell membrane dis-
turbing permeability and respiration functions of the cell

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,
in Combination with Antibiotics on Salmonella typhi 483
Figure 6. Zone of inhibition with silver nanoparticles.
Table 1. 1 ml sample + ampicillin and gentamycin [10 mcg]
[kept for 15 mins] and zone readings taken.
Zone for
sample +
ampicillin
Zone for
sample +
gentamicin
SR. No. Organism
P1 P2MEAN P1 P2MEAN
1. Salmonella
typhi 14 2017 13 1715
Plate 1 = P1; Plate 2 = P2.
[26]. Smaller AgNPs having the large surface area avail-
able for interaction would give more bactericidal effect
than the larger AgNPs [26]. It is also possible that A gNP s
not only interact with the surface of membrane, but can
also penetrate inside the bacteria [27]. Zone of inh ibition
test was done for identification of degree of inh ibition by
silver nanoparticles in combination with gentamicin and
ampicillin antibiotics. 1 ml of as-synthesized colloidal
solution of silver nanoparticles was taken with 10 mcg of
ampicillin and with 10 mcg of gentamicin for 15 minutes
separately. Zone readings were taken for both. The com-
parative study given in Table 1 show that the zone size
for sample with ampicillin for Salmonella typhi is larger
than the zone size for sample with gentamicin. Thus
sample is more effective in combination with ampicillin
for S. typhi in comparison to gentamicin.
4. Conclusion
Production of silver nanoparticles can be achieved through
different methods. Chemical approaches are the most po-
pular methods for the production. However chemical me-
thods cannot avoid use of toxic chemicals in synthesis
methods. There is a growing need to develop environ-
mentally friendly processes of nanoparticles synthesis
that do not use toxic chemicals. So the approach to elc-
trolytically deposit highly pure silver nanoparticles with
the plant extract like tea (containing antioxidant compo-
nents) as capping agent in present synthesis me- thod is
well justified.
Due to the antibiotic resistance developed by the bac-
teria it is very hard to manage different types of antibac-
terial drugs. There is great need of agents to kill bacteria
and other microorganisms [28]. Silver nanoparticles have
been reported to have antimicrobial activity against a
wide range of microorganism. The use of silver nanopar-
ticles antimicrobial effects are highly sought after be-
cause of its broad spectrum activity, high rate of effect-
tiveness, and low cost. Research is being done to find
superior forms of silver-based antimicrobial agents.
The combination effect of nanosilver and ampicillin
has more potential compared to the other antibiotics and
may be caused by both, the cell wall lysis action of the
ampicillin and the DNA binding action of the silver
nanoparticles (Fayaz et al., 2009). The antibiotic mole-
cules contain many active groups such as hydroxyl and
amino groups, which reacts easily with silver nano-
particles by chelation, for this reason, the synergistic ef-
fect may be caused by the bonding reaction with anti-
biotic and silver nanoparticles.
Therefore a study for the combination of silver nano-
particles with most practised antibiotics i.e. ampicillin
and gentamicin was undertaken with S. typhi which shows
that the combination of silver nanoparticles and ampicil-
lin is more effective than th e combination of silver nano-
particles and gentamicin for Salmonella typhi.
5. Acknowledgements
Author is thankful to the Director, M.A.N.I.T., Bhopal,
India for the financial support in the form of scholarship
and the facilities at the institute, RRCAT Indore for XRD
facility, HSADL, Bhopal for the TEM facility, Micro Bio
laboratory Mumbai for bactericidal studies.
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in Combination with Antibiotics on Salmonella typhi
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