METHODOLOGY

MATERIALS:

Chemicals Required:

 0.9% of Sodium chloride(NaCl )

 2.8% of Nutrient Agar(NA)
 1.3% Nutrient Broth(NB)
 Zinc Oxide nanopowder(ZnO)

Glass Materials:

 Petri-plates
 Conical flasks
 Test tubes
 Beaker
 Pipette
 Measuring cylinder

Instruments:

 Autoclave
 Laminar air flow hood
 Incubator
 Magnetic stirrer
 Pipette boy
 Micropipette
  Weighing balance
 Refrigerator

Miscellaneous:

 Spirit lamp
 Match box
 Cotton
 Mili Q Water
 magnetic beads

Culture:

24 hours E. coli culture

METHODS:

Preparation of 24 hours E. coli culture:

1 ml of [Link] culture from stock solution was added to 10 ml of

autoclaved Nutrient Broth and was kept in an incubator at 370c for 24 hours.

Preparation of saline water(0.9% of NaCl):

0.9g of NaCl was added in 100 ml of mili Q water.

Preparation of 2.8% Nutrient Agar:

2.8g of Nutrient Agar was added in 100 ml of mili Q water.

Preparation 1.3% Nutrient broth:

 1.3g of Nutrient Broth was added in 100ml of mili Q water.

Preparation of ZnO Nanoparticle stock solution:

0.3g of supplied ZnO nanoparticle was added in 100ml of autoclaved

Nutrient Broth with a magnetic bead under sterile condition.

Effect Of ZnO Nanoparticles On Microorganisms

Procedure-

1. 100ml of nutrient broth was prepared in two conical flasks with

magnetic beads.
2. 0.3gm of ZnO nanoparticle powder was added to one of the
conical flask and the other one is retained as positive control.
3. Both of the conical flasks were spiked with 100µl of fresh 24hr
[Link] culture and stirred for 2 hours and was stabilized.
4. After completion of stirring, serial dilution was carried out upto
10-9 concentrations from both ZnO treated and untreated flasks.
5. 100µl from 10-3,10-5,10-7 and 10-9 dilutions were taken from both
treated and untreated flasks and cultured by spread plate on
nutrient agar surface.
6. All the plates having dilution 10-3, 10-5, 10-7, 10-9 were kept in an
incubator at 370 c for overnight to see the inhibitory effect of ZnO
nanoparticle.
7. The number of colonies were counted and recorded.
 8. .

RESULT:(1)

Untreated(without nanoparticles) :

DILUTIONS NUMBER OF COLONIES OBTAINED

10-3 TNTC
10-5 386
10-7 9
10-9 2

Treated(ZnO(0.3g) in 100ml 0f Nutrient Broth )

DILUTIONS NUMBER OF COLONIES OBTAINED

10-3 1
10-5 3
10-7 0
10-9 0

(2)

After 2 hour incubation

Concentration Control ZnO Treated

10-3 TNTC 448
10-5 172 5
10-7 2 0
10-9 0 0
 After overnight incubation

SPREAD PLATE:

Concentration Control ZnO Treated

10-3 TNTC 1
10-5 815 0
10-7 18 0
10-9 0 0

POUR PLATE:

Concentration Control ZnO Treated

10-3 TNTC 30
10-5 TNTC 2
10-7 163 0
10-9 0 0

Other methods(Comparative growth of [Link] in different

concentration of ZnO nanoparticles):

 100ml of ZnO stock solution was prepared in a conical flask and

autoclaved.
 100ml of nutrient broth broth was prepared and autoclaved.
 After autoclaving different concentration of ZnO and nutrient broth
solution was prepared in test tubes a shown below in the table.
 CONCENTRATION VOLUME OF NUTRIENT VOLUME OF ZnO STOCK
BROTH
5mg 8.300ml 1.700ml
2mg 9.400ml 0.600ml
1mg 9.700ml 0.300µ
500µg 9.830ml 0.170µl
200µg 0.915ml 0.085µl
125µg 0.960ml 0.040µl
100µg 9.965ml 0.035µl
Control 10.00ml 0.000 µl

 All the above concentrations of ZnO and nutrient broth given above the
table were spiked with 100µl of 24hr [Link] culture and vortexed for
30secs.
 These test tubes were incubated at 370c for overnight.
 After incubation all the tubes were serially diluted to the concentrations of
10-2,10-4,10-6,10-8 (double serial dilution method).
 100µl of culture from all tubes were taken and cultured by spread plate
method on nutrient agar surface plates.
 All the plates were kept in an incubator at 37 0c for overnight to see the
inhibitory effect of ZnO nanoparticles on [Link].
 The number of colonies were counted and recorded.

TABULATION

concentration 10-2 10-4 10-6 10-8

 5mg 0190 0006 0001
2mg TNTC 0170 0002
1mg TNTC TNTC TNTC
500µ TNTC TNTC TNTC
200µ TNTC TNTC TNTC TNTC
125µ TNTC TNTC TNTC TNTC
100µ TNTC TNTC TNTC TNTC
control TNTC TNTC 0114 00O5

ABSTRACT

In this study, we investigate the antibacterial activity of ZnO nanoparticles with

various particle sizes. The products were characterized by x-ray diffraction (XRD)
analysis, transmission electron microscopy (TEM)and photoluminescence (PL)
spectroscopy. Bacteriological tests such as minimum inhibitory concentration
(MIC) and disk diffusion were performed in Luria-Bertani and nutrient agar media
on solid agar plates and in liquid broth systems using different concentrations of
ZnO by a standard microbial method for the first time. Our bacteriological study
showed the enhanced biocidal activity of ZnO nanoparticles compared with bulk
ZnO in repeated experiments. This demonstrated that the bactericidal efficacy of
ZnO nanoparticles increases with decreasing particle size. It is proposed that both
the abrasiveness and the surface oxygen species of ZnO nanoparticles promote
the biocidal properties of ZnO nanoparticles. To address the molecular basis of
ZnO nanoparticle action, a large set of genes involved in cell stress response,
motility, pathogenesis, and toxin production were selected for a gene expression
study. These results suggest that the antibacterial mechanism of ZnO
nanoparticles is most likely due to disruption of the cell membrane and oxidative
stress in [Link].
Discussion
Mashayekhi et al. studied the effect of metal oxide nanoparticles on bacteria. [46]
All nanoparticlesexcept ZnO showed toxicity to bacteria significantly more than
their micro counterparts. Nano ZnO toxicity to bacteria is reported at higher
concentrations (1,000 mg/L, Adams et al. [13]) than what was used in this study .
Gram positive and Gram negative bacteria did not behave differently to
nanoparticle toxicity. The order of observed toxicity to nanoparticles in [Link] was
Al2O3 <SiO2 >ZnO Different bacteria had varied tolerance to nanoparticles. TEM
images confirmed attachment of nanoparticles to the bacteria. Observed toxicity
of nanoparticles was not due to the free metal ions dissolved from metal oxides.
It seems that size of particles plays an important role in their [Link] et
al. also performed similar studies on bacteria: [47] E. coli cells, after contact with
ZnO, were damaged showing a Gram-negative triple membrane
disorganization. This behaviour causes the increase of membrane permeability
leading to accumulation of ZnO nanoparticles in the bacterial membrane and also
cellular internalization of these nanoparticles. Lower concentrations of ZnO
nanoparticles did not induce any damage E. Coli cells after contact with ZnO
nanoparticle concentrations higher than 1.3 x 10-3M and with some small
molecules and macromolecules in NA medium were damaged and the cell
contents may have leaked out. Cellular internalization of these nanoparticles was
observe Jones et al. reported on the antibacterial activity of ZnO nanoparticle
suspension on a broad spectrum of microorganisms. [49]. ZnO nanoparticles
showed a significant growth inhibition compared with the control. Significant

growth inhibition was detected with the usedexperimental setting for [Link].
For ZnO ultrafine powder and ZnO nanopowder, which have relatively large
particle sizes, reduced growth rates ( 50%) were observed. However, ZnO
nanoparticles, with smaller particle size, were able to reduce . 99% of growth coll
oidal suspension concentration of [Link] data for both sizes of ZnO
nanoparticles clearly suggest that the antibacterial activity of ZnO nanoparticles in
the dark is less than that in ambient laboratory conditions and the authors
concluded that the ambient laboratory conditions are sufficient for the optimal
biocidal activity of the ZnO nanoparticles, which is probably dependent on the
size of the [Link] et al. also performed a similar study using ZnO
nanofluids. [50] The results show that the ZnO nanofluids have bacteriostatic
activity against E. coli. The antibacterial activity increases with increasing
nanoparticle concentration and increases with decreasing particle size. Particle
concentration is observed to be more important than particle size under the
conditions of this [Link] analyses of the bacteria before and after treatment
with ZnO nanofluids show that the presence of ZnO nanoparticles damages the
membrane wall ofthe bacteri

Acknowledgement
It gives me great pleasure to acknowledge with thanks,my respectful and
beloved ones for helping me bring out my project report successfully.
First and foremost I would like to thank with immense
gratitude to probiosys management and Dr Danish sir for helping to undertake
this project work.
 I express my heart felt thanks to Mr satya narayan sahu my
father for being external guides and helping me avail the opportunity to carry out
this study.I would also like to thank the friends for extending their support and
cooperation.
I have immense pleasure in expressing my deep sense of
gratitude to Dr Danish for this valuable guidance,co-operation and support
without which this report would not have been complete.

CONCLUSION
We have demonstrated the enhanced bioactivity of ZnO nanoparticles by studying
the antimicrobial activity of suspensions with various concentrations using a
standard microbial method for the first time. The major mechanism through
which ZnO nanoparticles manifested antibacterial properties is by anchoring to
and penetrating the bacterial cell wall, and modulating cellular signalling by
dephosphorylating putative key peptide substrates on tyrosine residues. ZnO
nanoparticles act primarily in three ways against Gram-negative bacteria: (1)
nanoparticles mainly attach to the surface of the cell membrane and drastically
disturb its proper function, like permeability and respiration; (2) they are able to
penetrate inside the bacteria and cause further damage by possibly interacting
with sulfur- and phosphorus-containing compounds such as DNA;(3) nanoparticles
release Zn ions, which have an additional contribution to the bactericidal effect of
the ZnO nanoparticles . Although bacterial cell lysis could be one of the reasons
for the observed antibacterial property, nanoparticles also modulate the
phosphotyrosine profile of putative bacterial peptides, which could thus affect
bacterial signal transduction and inhibit the growth of the organisms. The effect is
dose dependent and is more pronounced against gram negative organisms than
gram-positive ones. The antibacterial effect of nanoparticles is independent of
acquisition of resistance by the bacteria against antibiotics. However, further
studies must be conducted to verify if the bacteria develop resistance towards the
nanoparticles and to examine cytotoxicity of nanoparticles towards human cells
before proposing theirtherapeutic use.

REFERENCES

1. Silver S, Phung LT. Bacterial heavy metal resistance: new surprises.

Annu Rev Microbiol 1996;50:753- 89.

2. M.N. Baker, J.M. Taras, America Water Works Association, Denver 1981.

3. [Link],nano:The essentials,McGraw Hill Publications,New York 2009