Mycosynthesis of silver nanoparticles by

optimizing physico-cultural conditions

and its antibacterial activity.

Under the guidance of

Mrs. Juhi Saxena Diksha Agarwal
Assistant professor (B.Sc Biotechnology part
Dr. B. Lal Institute of III )
biotechnology jaipur
 CONTENTS
• Introduction • Objectives
• Applications of
silver nano particles
• Method
• Scope of the study • Results
• Mechanism of • Future prospects
nanoparticle
synthesis • Summary
• Action of • References
nanopaticles
 INTRODUCTION
Nanotechnology is an emerging field in the area of interdisciplinary research, especially
in biotechnology (Natarajan, Selvaraj, & Ramachandra, 2010).

The branch of technology that deals with dimensions and tolerances of less than 100
nanometres, especially the manipulation of individual atoms and molecules.

Biogenic synthesis of nanoparticles provides advancement over chemical and physical

method as it is cost effective, environment friendly, easily scaled up for large scale
synthesis and in this method there is no need to use high pressure, energy, temperature and
toxic chemicals.
(Ingale et al 2013).

PLANT FUNGI BACTERIA

Fig 1:Biological components are involved in biosynthesis of nanoparticles
 APPLICATIONS OF
NANOTECHNOLOGY:

Fig 2: Application of nanotechnology (Arun G. Ingale  and A. N. Chaudhari 2013)

*
 SCOPE OF THE STUDY:
 The number of multi-resistant bacterial strains has been increasing at an
alarming rate and represents a major threat to modern medicine.
(Li et al, 2015)

 Therefore, development of or modification in antimicrobial compounds to

improve bactericidal potential is a priority area of research in the modern era
(Lara et al, 2010)

Fig 3: The challenge- Microbiological battle becomes tougher Abbreviations

C. difficile (Clostridium difficile ), CRE (Carbapenem-resistant Enterobacteriaceae ) ,
MDR Neisseria gonorrhoeae (Multidrug-resistant ),
ESBL(Extended-spectrum β lactamase producing Enterobacteriaceae),
MDR Salmonella (Multidrug-resistant ), MRSA ( Methicillin-resistant Staphylococcus aureus ),
MDR Pseudomonas (Multidrug-resistant )
 Different types of biogenic metal nanoparticles are
produced using green chemistry like copper, zinc,
titanium, magnesium, gold, alginate, and silver; of
them silver nanoparticles (AgNPs) have proved to be
most effective as they have good antimicrobial
efficacy against bacteria, viruses, and other
eukaryotic microorganisms .

Fig 4 : Biogenic nanoparticles against multidrug pathogens

MECHANISM OF NANOPARTCLE SYNTHESIS:

Fig 6: Bio reduction process

Fig 5: Flowchart showing the biosynthesis of nanoparticles

(Prathna, T. C., et al. 2010)
ACTION OF NANOPARTICLES:

Franci, Gianluigi, et al. 2015

Fig 7: Action of silver nanoparticles

 OBJECTIVES:
• Synthesis of silver nanoparticles.
• Characterization of synthesized silver nanoparticles.
• Optimization of physico-cultural conditions on biological synthesis of
silver nanoparticles.
• Evaluation of antibacterial activity against Extended Spectrum beta
Lactamase E. coli bacteria.
 METHODS:
I. CULTURAL/MACROSCOPIC AND MICROSCOPIC IDENTIFICATION OF
SELECTED FUNGI BY LACTO PHENOL BLUE(LCB) STAINING METHOD.

Take a clean slide and place a drop of lacto phenol cotton blue in centre of
slide.

Then cut the tape and hold the cut piece with the help of forcep.

Place the tape onto the surface of the fungal colony, where sporulation is
visible.

Pull away gently the tap from the colony and place it on the drop of lacto
phenol cotton blue.

Place cover slip on it.

Observe under microscope

II: SYNTHESIS OF METAL NANOPARTICLE FROM FUNGAL ISOLATES:

Fungi were inoculated in a flask containing production broth. (28 oC for 5-6 days, 120
rpm).

Mycelia filtered through Whatman filter paper and was inoculated (fresh weight) in
sterilized distilled water followed by incubation at 28 oC for 24 hours.

Cell filtrate was treated with metal ions solution and incubate at room temperature.

Fig 8: Synthesis of metal nanoparticles from fungal isolate (Sastry et al. 2003 and
ahmed et al. 2002)
III. CHARACTERIZATION OF SYNTHESIZED
NANOPARTICLES

Color change of fungal cell filtrate indicates the formation of metal

nanoparticles.

Characterization was done with UV-Visible spectrophotometer.

Synthesis show absorbance peak around , which is specific for the metal
nanoparticles.

Fig 9: Surface plasmon resonance (By Frédéric Pelletier* and Bénédicte Thiébaut)

 IV. OPTIMIZATION OF PHYSICO- CULTURAL CONDITIONS ON BIOLOGICAL SYNTHESIS OF SILVER NANOPARTICLES

Table no. 1: showing different optimization parameters

 V. ANTIBACTERIAL ASSAY
Disc diffusion assay was carried out to evaluate antibacterial activity against clinical
isolate of ESBL E. coli (Provided by Dr. B. Lal Clinical Laboratory Pvt. Ltd) (KDC-233 ESBL E.coli culture)

Inoculum of bacteria was developed by growing organism overnight in Muller Hinton

liquid medium in shaker incubator at 370C.

Using sterile swabs bacteria was inoculated on autoclaved Muller Hinton agar plate.

Plates were allowed to set and then sterile paper discs impregnated with nanoparticles
were placed using sterile forceps for disc diffusion.

Plates were incubated at 37oC and after 24 hrs, zone of inhibition was measured.

(Positive control- Cefotaxime + clavulanic acid, Negative control- Fungal filtrate was
also be maintained)
(Birla et al 2009, Gade et al 2008, Naqvi et al 2013, Subhashini et al 2014)
 FUTURE PROSPECTS:
1. Further studies arerequired for determining anti fungal activity of
nanoparticles.
2. Molecular characterization of fungal isolate.

SUMMARY:
1. The biosynthesis of nanoparticles is clean, nontoxic, and
environmentally acceptable “green chemistry”
2. For rapid and maximum yield of silver nanoparticles
optimization process with different physico cultural conditions
is required which improve its antibacterial efficiency.
3 Silver nanoparticles are used as effective antimicrobial agents.
They have bactericidal potential against multidrug-resistant
bacteria organisms.
 REFERENCES:
Gong, P., Li, H., He, X., Wang, K., Hu, J., Tan, W., ... & Yang, X. (2007). Preparation and
antibacterial activity of Fe3O4@ Ag nanoparticles. Nanotechnology, 18(28), 285604.
Lara, H. H., Ayala-Núnez, N. V., Turrent, L. D. C. I., & Padilla, C. R. (2010). Bactericidal
effect of silver nanoparticles against multidrug-resistant bacteria. World Journal of Microbiology
and Biotechnology, 26(4), 615-621.
Li, X. Z., Plesiat, P., & Nikaido, H. (2015). The challenge of efflux-mediated antibiotic
resistance in Gram-negative bacteria. Clinical microbiology reviews, 28(2), 337-418.
Prathna, T. C., Mukherjee, A., Raichur, A. M., Mathew, L., & Chandrasekaran, N. (2010).
Biomimetic synthesis of nanoparticles: science, technology & applicability. INTECH Open
Access Publisher.
Saxena, J., Sharma, M. M., Gupta, S., & Singh, A. (2014). Emerging role of fungi in
nanoparticle synthesis and their applications. World Journal of Pharmacy and Pharmaceutical
Sciences, 3(9), 1586-1613.
Singh A., Sharma M. M and Batra A. (2013) Synthesis of gold nanoparticles using chick pea
leaf extract using green chemistry. Journal of Optoelectronics and Biomedical Materials 5: 27 –
32.
Katrin Quester Miguel Avalos-Borja, Alfredo Rafael Vilchis- Nestor Marco Antonio Camacho-
López,Ernestina Castro-Longoria (2013): SERS Properties of Different Sized and Shaped Gold
Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa
Extract , plos one October 2013 | Volume 8 | Issue 10 | e77486 ,2013.
Mahendra Rai, Alka Yadav, Paul Bridge and Aniket Gade(2009): Myconanotechnology: a New
and Emerging Science ,CAB International 2009
Arun G. Ingale and A. N. Chaudhari(2013) : Biogenic Synthesis of Nanoparticles and
Potential Applications: An Eco-Friendly Approach, J Nanomed Nanotechol 2013
 Achievements

 Second Prize in Oral Presentation: Synthesis of mycogenic nanoparticles

and their antibacterial activity in Biochrome-2015 Annual Techno-Cultural Fest
at Dr. B. Lal Institute of Biotechnology on 15th – 17th October.
 Second Prize in Poster Presentation: Mycosynthesis of nanoparticles
from fungal isolate and its antibacterial activity in Biochrome 2015,
Annual Techno-Cultural Fest at Dr. B. Lal Institute of Biotechnology on 15th –
17th October.
 Poster Presentation: Biomimetics: Route for nano biosynthesis with
biomedical applications in National Seminar on “Advances in natural science
for indigenous development in India” at Department of Botany-ISCA,
University of Rajasthan, Jaipur on 30th –2nd October, 2015.