THE BACTERICIDAL EFFECT OF SILVER
NANOPARTICLES
Jaynesh Shah
Greg Pudewell
Edwin L. Youmsi Pete
John Pack
OUTLINE
Introduction
 Methods
 Results and Discussions
 Conclusions

PROBLEM?
Strains of bacteria
resistant to current
antibiotics
 Strong incentives to
develop new bactericides
 Bactericidal
nanomaterials could be
extremely helpful and
are being pursued in a
timely manner

http://wiki.sis.edu.hk/users/12leungrj2/
MEMBRANE STRUCTURES

Bacteria have different
membrane structures


Gram-negative


Based on organization of key
component, peptidoglycan, in
the membrane
Thin peptidoglycan layer (~2-3
nm) between the cytoplasmic
membrane and the outer
membrane
Gram-positive

No outer membrane, but have
a thick peptidoglycan layer
(~30 nm)
http://silverfalls.k12.or.us/staff/read_s
hari/mysite/chapter_24_AB.htm
SILVER BASED COMPOUNDS


Silver is known to exhibit a strong
toxicity to a wide rang of microorganisms.
Extensive use in many bactericidal
applications



http://www.shutterstock.com/pic3609757/stock-photo-silversymbol.html
Inorganic composites with a slow silver
release rate used as preservatives
Compounds composed of silica gel
microspheres containing a silver
thiosulfate complex are mixed into
plastic for long lasting antibacterial
protection
Silver compounds used in the medical
field to treat burns and a variety of
infections.
http://www.los-angelesinjury-lawyerblog.com/2008/03/california
_burn_lawyer_acciden.html
BACTERICIDAL EFFECTS AND MECHANISM


Bactericidal effect of silver
ions on micro-organisms is
well known, but the
mechanism is only
partially understood.
Proposed theory describes
that ionic silver strongly
interacts with thiol groups
of vital enzymes and
inactivates them.
http://www.advancedagrotech.com
/a/pr_nanopaints.htm
PREVIOUS RESEARCH


DNA loses its replication
ability once the bacteria
have been treated with
silver ions
Evidence of structural
chances in the cell
membrane as well as
the formation of small
electron-dense granules
formed by silver and
sulfur
http://www.pbs.org/wgbh/nova/sciencenow/
3214/01-coll-04.html
CHANGES WITH NANOTECHNOLOGY


Physical properties of
metal particles at the
nanoscale differ from
those of the ion and the
bulk material.
Particles exhibit
remarkable properties
such as increased
catalytic activity due to
morphologies with
highly active facets.
http://www.anapro.com/english/product
/nano-silver-paste.asp
EXPERMENTAL PROCEDURE

Silver is tested with four types
of Gram-negative bacteria





E. coli
V. cholera
P. aeruginosa
S. typhus
Apply several electron
microscopy techniques to
study the mechanism by
which silver nanoparticles
interact with these bacteria
http://www.biologie.uni-hamburg.de/bonline/e03/03e.htm
PREPARATION OF SILVER NANOPARTICLES




The silver nanoparticles used in this
work were synthesized by
Nanotechnologies, Inc.
The final product is a powder of silver
nanoparticles inside a carbon matrix,
which prevents coalescence during
synthesis.
The silver nanoparticle powder is
suspended in water in order to perform
the interaction of the silver nanoparticles
with the bacteria; for homogenization of
the suspension a Cole-Parmer 8891
ultrasonic cleaner (UC) is used.
The particles in solution are
characterized by placing a drop of the
homogeneous suspension in a
transmission electron microscope (TEM)
copper grid with a lacy carbon film and
then using a JEOL 2010-F TEM at an
accelerating voltage of 200 kV.
FIRST STEP: TESTING OF SILVER
NANOPARTICLES


As a first step, several
concentrations of silver
nanoparticles (0, 25, 50, 75 and
100 μg ml−1) were tested against
each type of bacteria.
Agar plates from a solution of agar,
Luria–Bertani (LB) medium broth
and the different concentrations of
silver nanoparticles were prepared,
followed by the plating of a 10 μl
sample of a log phase culture with
an optical density of 0.5 at 595 nm
and 37 ◦C.
INTERACTION OF BACTERIA WITH SILVER
NANOPARTICLES





The interaction with silver nanoparticles was
analysed by growing each of the bacteria to a log
phase at an optical density at 595 nm of
approximately 0.5 at 37 ◦C in LB culture medium.
Then, silver nanoparticles were added to the
solution, making a homogeneous suspension of
100 μg ml−1 and leaving the bacteria to grow for
30 min.
The cells are collected by centrifugation (3000
rpm, 5 min, 4 ◦C), washed and then resuspended
with a PBS buffer solution.
A 10 μl sample drop was deposited on TEM copper
grids with a lacy carbon film and the grid was then
exposed to glutaraldehyde vapours for 3 h in order
to fix the bacterial sample.
The bacteria were analysed in a JEOL 2010-F TEM
equipped with an Oxford EDS unit at an
accelerating voltage of 200 kV in scanning mode
using the HAADF detector, in order to determine
the distribution and location of the silver
nanoparticles, as well as the morphology of the
bacteria after the treatment with silver
nanoparticles.
ALTERNATIVE SAMPLE PREPARATION
TECHNIQUE






In order to have a more profound understanding of the bactericidal mechanism of the
silver nanoparticles, a different sample preparation technique was prepared
E. coli samples, previously exposed to silver nanoparticles, following the same
procedure of interaction mentioned previously, were then fixed by exposure to a 2.5%
glutaraldehyde solution in PBS for 30 min, followed by a dehydration of the cells using
a series of 50, 60, 80, 90 and 100% ethanol/PBS solutions and exposing the sample
for ten minutes to each solution in increasing order of ethanol concentration
The cells were finally embedded into Spurr resin and left to polymerize in an oven at
60 ◦C for 24 h.
The polymerized samples were sectioned in slices of thickness of ∼60 nm.
We were then able to analyse the interior of the bacteria in the TEM in STEM mode.
The same procedure but with 100 μg ml−1 of ionic silver, from a 1 mM solution of
AgNO3, was performed to compare effects of silver in ionic and nanoparticle form.
TEM ANALYSIS USING SAMPLE STAINING
 TEM analysis using sample staining was
also carried out.
 The sample preparation followed the same
procedure as the cross-sectioned sample
slices but before the dehydration process
the cells were tinted with a 2%
OsO4/cacodylate buffer for 1 h.
 These samples were analysed in a JEOL
2000 at an accelerating voltage of 100 kV.
ANALYSIS OF THE ELECTROCHEMICAL
BEHAVIOUR OF SILVER NANOPARTICLES





The electrochemical behaviour of silver nanoparticles in water
solution was also analysed.
Stripping voltammetry of silver nanoparticles, in dissolution in an
electrolyte solution, was carried out using a 25 μm diameter
platinum ultramicroelectrode.
To detect silver (I) electrochemically at low concentrations, it is
necessary to electro-deposit silver onto the electrode surface in a
pre-concentration step by holding the potential of the electrode at
−0.3 V versus Ag/AgCl for 60 s
This procedure reduces Ag+ to Ag0, which plates onto the electrode
surface.
When the potential is swept positively from −0.3 to +0.35 V, the
deposited silver is oxidized to Ag+ and stripped from the electrode,
giving a characteristic stripping peak with a height proportional to
the concentration of Ag+ in the solution.
STRIPPING VOLTAMMETRY
RESULTS



TEM analysis shows
silver particles
agglomerated inside
the carbon matrix
a) illustrates the
agglomeration
b)-d) most common
morphologies of the
particles used are
cuboctahedral,
icosahedral, and
decahedral
RESULTS





Each type of bacteria
required different
concentrations of silver
Upper left, E. coli
Upper right, S. typhus
Bottom left, P.
aeruginosa
Bottom right, V.
cholerae
RESULTS
Nanoparticles are found on the cell membrane
as well as inside the bacteria
 Cells on the membrane and the interior are the
same size
 Particles that interact with the membrane is
faceted, most likely decahedral
 Mean size of interacting particle was 5 nm
 Suggests that bactericidal effect of particles is
size dependent

RESULTS
a)
b)
c)
d)
e)
Shows presence of silver
nanoparticles in membrane
of E. coli
Close up of E. coli
membrane
Close up of E. coli interior
E. coli treated with silver
nitrate
Shows silver ions
immediately released at
concentration of 1
micromolarity
RESULTS

Results lead to three hypotheses





Nanoparticles smaller than 10 nm interact with bacteria
Nanoparticles are spherical
Amount of carbon in the sample can be ignored
Weight percent of nanoparticles between 1 and 10
nm makes up only 0.093% of the sample
This seems small but concentrations over 75
micrograms/ml eliminated all bacterial growth

0.093% corresponds to more than enough
nanoparticles
RESULTS
a)
b)
c)
d)
Silver particles
can be seen in S.
typhus
Intensity profile
of localized
region in a)
Morphology
distribution
Size distribution
RESULTS
Silver nanoparticles affect alterations in the
membrane of the bacteria
 Nanoparticles may react with DNA
 Also affect the cell in areas such as the
respiratory chain and cell division, ultimately
resulting in cell death
 Possible electrostatic interaction between
charged particles and charged bacterial wall

CONCLUSIONS

Silver nanoparticles act in
three ways against gramnegative bacteria.



Attach to the surface of the
cell membrane perturb its
proper function
Penetrate inside the
bacteria and cause damage
Release silver ions, which
will have an additional
contribution to its
bactericidal effect
http://farm1.static.flickr.com/137/355583438_3667e9ef
f0.jpg
CONCLUSIONS


Metallic nanoparticles
have antibacterial
properties and exhibit
increased chemical
activity.
This is due to their large
surface to volume ratios
and crystallographic
surface structure.
http://wwwche.engr.ccny.cuny.edu/ilona/public/asymm_silver.jpg
CONCLUSIONS

http://science-hub.com/wpcontent/uploads/2009/06/nanoparticles1.jpeg
High angle annular dark
field (HAADF) scanning
transmission electron
microscopy (STEM) has
been proven to be a very
useful in the study of
bactericidal effects of
silver particles
FURTHER RESEARCH
Application of HAADF-STEM
should be extended to other
related research
 Effect of electrostatic forces
on interaction of the
nanoparticles with bacteria
should be further studied.
 Understand the formation of
low density region formation is
a mechanism of defense

http://www.nanotechnologyinvesting.us/nanotechnolog
y-robot-530.jpg