Marougail-Pushani 1

Introduction

For centuries, silver has been used as an antibacterial agent. Ancient Greeks and

Romans used silver to disinfect their food and water, and other pioneers even submerged

silver coins in their drinks to keep them fresh. By the 1920s, the United States Food and

Drug Administration approved silver as a type of antibacterial agent (Siauw, Winnie).

Today, nanotechnology has produced revolutionary results across science and medicine.

Silver nanoparticles have been a product of such revolutionary findings.

The emergence of new nanotechnologies has proven to be a solution in many

environmental and technological challenges. The synthesis of silver nanoparticles is

beginning to play a greater role in nanomedicine due to their potential microbial

properties toward pathogenic microbes, especially in a time where antibacterial resistance

is prevalent. Antimicrobial resistance is an increasing problem in the treatment of

bacterial infections. The widespread use and overuse of broad-spectrum antibiotics has

led to antimicrobial resistance to antibiotics for many bacterial pathogens. However,

nowadays, the emergence of new nanotechnologies has deemed silver nanoparticles a

viable alternative to antibiotics that potentially solves the problem of antibacterial

resistance.

Due to their antimicrobial properties, silver nanoparticles are implemented

throughout many everyday-products, such as hair straighteners and baby-changing tables,

in order to inhibit the growth of any bacteria. Such bacteria include Escherichia coli, a

gram negative bacterium. As aforementioned, E. coli, like other bacteria, has the ability

to resist antibiotics and other treatments. Although E. coli was the only bacterium used

while researching, the results from this experiment could be used to prevent the growth of
 Marougail-Pushani 2

various antibiotic-resistant bacteria in many settings, such as certain hospital settings,

meat-handling facilities, and in nanoparticle-impregnated products.

Discovering methods to prevent antibiotic-resistance can aid in the continuous

battle against bacteria. If these antibiotic-resistant bacteria are not addressed and treated

effectively, they may continue to harm humans. In this experiment. two different silver

nanoparticle solutions were created using 1.0 M silver nitrate, AgNO3 solution and either

trisodium citrate dihydrate or Camellia sinensis as a reducing agent to synthesize the

nanoparticles. The two different stabilizing and reducing agents were impregnated into

agar, which helped to determine which synthesis of nanoparticles would most effectively

stunt the growth of the E. coli.

The results of this experiment can determine whether green or chemically

synthesized silver nanoparticles would most effectively inhibit bacterial growth. Based

on experimental results, the method of green synthesis, using Camellia sinensis as a

reducing agent to synthesize the nanoparticles, was more effective in inhibiting bacterial

growth.

This research would allow manufacturers that impregnate products with the

nanoparticles to develop an efficient way to create a large quantity of the nanoparticles in

a non-toxic, environmentally-friendly way. Finding methods that prevent antibiotic

resistance could lead to revolutionary results in medicine and nanotechnology to better

the health and lives of human beings.

 Marougail-Pushani 3

Review of Literature

The emergence of nanoscience and new nanotechnologies has proven to be a

solution in many environmental and technological challenges. The synthesis of silver

nanoparticles is beginning to play a greater role in nanomedicine due to their potential

microbial properties toward pathogenic microbes, especially in a time when antibacterial

resistance is prevalent. Antimicrobial resistance, the ability of bacterial and other

microorganisms to resist the effects of an antibiotic to which they were once sensitive to,

is an increasing problem in the treatment of bacterial infections (Siauw, Winnie). The

widespread use and overuse of broad-spectrum antibiotics has led to antimicrobial

resistance to antibiotics for many bacterial pathogens. However, nowadays, the

emergence of nanoscience and new nanotechnologies has deemed silver nanoparticles a

viable alternative to antibiotics that potentially solves the problem of antibacterial

resistance (Franci, Gianluigi, et al).

Silver nanoparticles are nanoparticles of silver between 1 nanometer and 100

nanometers in size. These nanoparticles contain a large percentage of silver oxide. Silver

nanoparticles have attracted greater interest than other nanoparticles due to their unique

physical, chemical, and biological properties. Distinct chemical properties of silver

nanoparticles include high electrical and thermal conductivity, surface-enhanced Raman

scattering, chemical stability, catalytic activity, and nonlinear optical behavior; such

properties make silver nanoparticles valuable in microelectronics and medical imaging

(Tran, Quang Huy, et al). Silver nanoparticles are most notably known for their

antimicrobial capability; they are reported to possess antifungal, anti-inflammatory,

antiviral, anti-angiogenesis (inhibits the growth of new blood vessels), and antiplatelet
 Marougail-Pushani 4

activity (inhibits blood clot formation) (Tran, Quang Huy, et al). For this reason, silver

nanoparticles have been used to prevent and treat various diseases, specifically, infectious

diseases.

As aforementioned, nanoparticles range from 1 to 100 nanometers in size. When

it comes to a silver nanoparticle, one nanoparticle is essentially ineffective, however

colloids of billions and trillions of nanoparticles are profoundly more effective when it

comes to their antimicrobial properties and inhibiting the growth of bacteria. These

nanoparticles attack bacteria in quite specific ways: respiration, replication, and cell wall

destruction. Bacteria are approximately 1000 nanometers in size. They use enzymes that

act as catalysts for energy, oxygen, and nutrient metabolism in order to replicate. Bacteria

depend on an enzyme to metabolize oxygen to live. However, in order to disrupt this

exponential replication, the silver ions disrupt the effectiveness of the bacterial enzymes

and thus their energy and oxygen metabolism uptake, killing them (Siauw, Winnie).

Additionally, these silver ions can also disrupt the bacterial replication process by

deterring their DNA backbone; this occurs by a mechanism of toxicity which involves the

disruption of the mitochondrial respiratory chain by the silver nanoparticles, which leads

to the production of ROS (Reactive Oxygen Species) and the interruption of ATP

synthesis, which in turn causes the DNA damage (Huijing Bao, et al). Finally, silver ions

destroy a microbe by binding to the cell wall of the bacteria in order to weaken the

structure of the cell and thus speed up the process of bursting the bacteria. Therefore,

silver ions prevent the growth of bacteria through a potent defense system by disrupting

the replication and respiratory process of the microbes (Siauw, Winnie).

 Marougail-Pushani 5

Figure 1. How Silver Nanoparticles Kill Bacteria (Siauw, Winnie)

Figure 1 displays a bacterial cell interacting with silver nanoparticles. As seen

above, the silver ions bind to the cell and destroy the microbe through the processes

described above.

This research experiment examined the ability of the silver nanoparticles to attack

and destroy bacteria by testing it against a common bacterium, Escherichia coli. E. coli is

a prevalent bacterium living in the intestines of warm-blooded organisms, including

humans. E. coli can also be found in the environment and in intoxicated food and water.

Several strains of E. coli exist. E. coli consists of a diverse group of bacteria. Although

many genetic variants of E. coli are harmless, certain strains of E. coli are pathogenic.

Pathogenic E. coli strains can cause kidney failure, urinary tract infections, respiratory

illnesses, pneumonia, and diarrhea (Siauw, Winnie).

Prior to recent technological advancements, such as the Scanning Electron

Microscope (SEM) and Transmission Electron Microscope (TEM), scientists were

limited in studying nanoparticles due to their inability in determining their size and shape.

A recent study by Sukdeb Pal, Yu Kyung Tak, and Joon Myong Song, from the Research

Institute of Pharmaceutical Sciences and College of Pharmacy in Seoul National

University, South Korea investigated the antibacterial properties of differently shaped

silver particles against Escherichia coli, both in liquid systems and agar plates. Truncated
 Marougail-Pushani 6

triangular silver nanoplates with a lattice plane as the basal plane displayed the strongest

biocidal action, compared to spherical and rod-shaped nanoparticles. It is believed that

the nanoparticle size and the presence of a plane combine to promote this biocidal

property. Smaller-sized nanoparticles and more triangular-shaped nanoparticles make it

easier for the particles to bind to the bacterium and ultimately destroy it; a triangular,

sharper, shape provides a more efficient way to burst and attack the cell wall of that

certain microbe. The results of this experiment demonstrated that silver nanoparticles

experience a shape and size dependent interaction with the gram-negative organism, E.

coli (Pal, Sukdeb, et al).

Silver nanoparticles are synthesized through several different methods, including

green synthesis and chemical synthesis methods, which fall into larger categories of

bottom-to-up and top-to-bottom synthesis, respectively. Green synthesis is the least

common methodology of creating these nanoparticles, however in recent times, it has

been investigated in order to find an eco-friendly and nontoxic technique for production

of well-characterized nanoparticles. What has been tested to be the most suitable for the

production of a large-scale biosynthesis of metal nanoparticles is plants. Nanoparticles

produced by plants are more stable and create these metal nanoparticles at a faster rate

(Siavash Iravani).
 Marougail-Pushani 7

Figure 2. Types of Synthesis of Nanoparticles (Ahmed, Shakeel)

Figure 2 displays the different methods used to synthesize metal nanoparticles.

Through a green synthesis method, nanoparticles are created by the reduction and

stabilization of silver ions by combination of biomolecules such as polysaccharides and

polyphenols, which are present in plant extracts and are environmentally benign.

Polyphenols are compounds containing more than one phenolic hydroxyl group (Ahmed,

Shakeel, et al). Polysaccharides are complex carbohydrates constituted of

monosaccharides joined together by glycosidic bonds and are often one of the main

structural elements of plants ("Polysaccharide | Definition of Polysaccharide by Medical

Dictionary").
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Figure 3. Polysaccharide (Foist, Laura)

Figure 3 illustrates the structure of a polysaccharide, which is used in the green

method of synthesizing nanoparticles.

Figure 4. Polyphenols (Klub, Le)

Figure 4 illustrates an example of the structure of a polyphenol, since various

polyphenols exist, which are also used in the green method of synthesizing nanoparticles.

Many plants are used in the synthesis of metal nanoparticles such as Camellia

sinensis, green tea. Plants are used as reducing agents, which bring about reduction and

are oxidized by losing electrons. Meanwhile, capping agents allow for the stabilization of

nanoparticles, from their surface to their growth (Neena, D, et al). Green tea leaf extract

has proven to be effective in the production of nanoparticles and in their fight against

bacteria. Green tea extract acts as both a reducing agent and a capping agent through its

use of polysaccharides and polyphenols and flavonoids, producing silver nanoparticles at

 Marougail-Pushani 9

about 20-90 nanometers with a crystalline structure and spherical shape; they are also

quite effective against the growth of E. coli. (Ahmed, Shakeel, et al).

Figure 5. Green Synthesis of AgNPs (Kumar, Rahul)

Figure 5 displays how silver nanoparticles are created using leaf extract. The

reducing agent is simply added to a solution of silver nitrate and stirred until the solution

changes color and the nanoparticles are formed.

Figure 6. Inside the Green Synthesis Method (Kumar, Rahul)

Figure 6 displays a simple diagram illustrating how through green synthesis, the

plant extract (which acts as a reducing and capping agent) phytosynthesizes with a metal

solution to reduce metal ions using its natural makeup (such as polyphenols) to create the

metal nanoparticles.

Besides the eco-friendly use of a green reduction to produce silver nanoparticles,

chemical reduction is the most common scheme for syntheses of silver nanoparticles.

Different organic and inorganic reducing agents, such as sodium borohydride (NaBH4)

and sodium citrate (Na3C6H5O7) are used for the reduction of silver ions (Ag+) in aqueous

solutions; capping agents are also used to stabilize the surface and growth of the

nanoparticles. The reason chemical reduction is the most common use of synthesizing

metal nanoparticles is because they produce a large quantity of nanoparticles that can be
 Marougail-Pushani 10

synthesized in a short span of time. However, in this type of synthesis, the chemicals used

are toxic and lead to non-eco-friendly by-products. The method of using sodium citrate as

a reducing agent is derived by J. Turkevich, who named it the Turkevich method

(Ahmed, Shakeel, et al).

Figure 7. Trisodium Citrate Dihydrate (Chemical Book)

Figure 7 displays the structure of trisodium citrate dihydrate, also called citric

acid. Sodium citrate is also composed of three carboxylate groups ("Sodium Citrate: A

Universal Reducing Agent).

AgNPs

Figure 8. Inside the Chemical Synthesis Method (Lacson, Gene)

Figure 8 displays how silver nitrate provides Ag+ ions that are reduced by sodium

citrate, which itself becomes oxidized. The excess sodium citrate present in the solution

caps or stabilizes the particles through the carboxylate groups and prevents them from

growing as larger particles, thus acting as both a reducing and capping agent, and

successfully producing silver nanoparticles (Lacson, Gene).

 Marougail-Pushani 11

Other researchers have previously conducted experiments regarding the synthesis

of silver nanoparticles. In one experiment by Shakeel Ahmed, Saifullah, Mudasir Ahmad,

Babu Lal Swami, and Saiqa Ikram, researchers at the Department of Chemistry, Faculty

of Natural Sciences in New Delhi, India, silver nanoparticles were synthesized using the

green synthesis method and aqueous leaf extract Azadirachta indica was used as the

reducing agent. The silver nanoparticles showed antibacterial activities in both

Staphylococcus aureus and Escherichia coli microorganisms. The antibacterial activity

was measured based on the inhibition zone around the disc impregnated with plant

extract and synthesized silver nanoparticles (Ahmed, Shakeel, et al). Later, various tests

were conducted on the silver nanoparticles. One test was used to determine the size of the

nanoparticles, and another test was used with the different concentrations of the

nanoparticles and varying leaf extract solutions to measure their absorbance. The

researchers concluded that the Azadirachta indica was both effective and efficient in the

use of silver nanoparticles (Ahmed, Shakeel, et al). The experiment of these predecessors

is similar to this chemistry experiment that was conducted, as it uses the green method of

silver nanoparticle synthesis. However, in contrast, leaf extract Azadirachta indica was

used as a reducing agent while this experiment used green tea leaf extract as the reducing

agent. Moreover, the previous research tested the synthesized silver nanoparticles on their

antibacterial activities in both Staphylococcus aureus and Escherichia coli

microorganisms, while this research experiment only tested the nanoparticles against E.

coli. Finally, while this research experiment focused on the antimicrobial activities of the

nanoparticles, the previous research also tested the nanoparticles for their properties of

size and absorbance.

 Marougail-Pushani 12

Another research experiment by Pham Van Dong, Chu Hoang Ha, Le Tran Binh,

and Jrn Kasbohm affiliated with Vietnam National University, the University of

Greifswald, and VAST, examined the different chemically synthesized AgNPs using

polyvinylpyrrolidone, sodium citrate, hydrogen peroxide, and sodium borohydride.

Triangular and spherical nanoparticles were created and the difference in morphologies

was tested against the growth of Escherichia coli. The morphologies (size and shape) and

structures of the nanoparticles were characterized by transmission electron microscopy,

UV-visible spectroscopy, and X-ray diffraction. The research and analysis conclude that

the triangular AgNPs were more effective in their antimicrobial properties in comparison

to the spherical AgNPs. The research concluded that morphology plays a vital role in the

effect silver nanoparticles have on bacteria (Dong, Pham Van et al). The previous

research resembles this experiment, as it uses chemical methods and sodium citrate as a

reducing agent to synthesize the silver nanoparticles. However, the previous research

focuses on the morphology of the nanoparticles and how the morphology plays a role in

the effectiveness in inhibiting the growth of bacteria. This previous research remains

relevant to this research experiment because it introduces a property of the nanoparticles

that affects their antimicrobial activity and it allows for the consideration of the

morphology as an uncontrolled variable in the experiment.

Yet another previous experiment by Yan Zhou, Ying Kong, Subrata Kundu,

Jeffrey D. Cirillo, and Hong Liang at Texas A&M University investigated tuberculosis

(TB). The purpose of this study was to examine the effects of gold and silver

nanoparticles against Escherichia coli. The main intention was to analyze some sort of

relationship between nanoparticles and a potential anti-TB drug. Once the silver NPs
 Marougail-Pushani 13

were synthesized, their morphology was characterized using transmission electron

microscopy (TEM). As another factor, the research also included different concentrations

of NPs that were applied to the bacterial culture. The way the effect of the NPs was

determined was through the independent variable of counting colonies (CFU). The effect

between NPs and bacteria was also analyzed through scanning electron microscopy, and

the antibacterial effects of the nanoparticles on the bacteria were determined by recording

fluorescent protein expression levels. The results suggested that nanoparticles do have

great antibacterial effects and could be potentially used at an anti-TB compound (Zhou,

Yan et al). The previous experiment that was just described remains relevant to this

experiment because both determined the effect of the nanoparticles by counting colonies.

However, in contrast, the previous experiment tested varying concentrations of

nanoparticles against the E. coli.

Silver nanoparticles possess several properties that make them useful in todays

economy. They can be synthesized through various methods, such as using plant extract

as a reducing agent (green synthesis) and using sodium citrate as a reducing agent

(chemical synthesis). Both methods have their advantages and disadvantages and this

experiment investigates which is most effective in inhibiting the growth of E. coli, which

was influenced by previous research mentioned above.

 Marougail-Pushani 14

Problem Statement

Problem:

The purpose of this experiment was to determine which method of synthesizing

silver nanoparticles (green vs. chemical) would yield the greatest effect in inhibiting the

growth of Escherichia coli.

Hypothesis:

The green synthesized silver nanoparticles will result in the smallest percentage

growth of Escherichia coli.

Data Measured:

In this experiment, the independent variables were the two different synthesized

silver nanoparticles. The percentage growth of bacteria in the Petri dish was the

dependent variable measured. This was measured by using grid paper and counting the

bacterial growth in each square. A detailed explanation of this process is located in the

Data and Observations. The controls of the experiment included: positive, negative, green

synthesis, and chemical synthesis. The constants of this experiment were the incubation

temperature (37C) and the concentration of the silver nitrate solution (1.0 M) used to

prepare the nanoparticles. Eight trials of the green synthesized nanoparticles and eight

trials of the chemically synthesized nanoparticles were conducted. The controls were run

every day for a total of four trials of each control. Descriptive statistics were used to

analyze the effects of the different silver nanoparticles on the growth of Escherichia coli.
 Marougail-Pushani 15

Experimental Design

Materials:

0.17 g Silver Nitrate, AgNO3 Bchner Funnel

1.47 g Sodium Citrate, Na3C6H5O7 Vacuum Suction
16.0 g Camellia sinensis Leaf Extract (1) Scoopula
23 grams Dehydrated Agar (2) Weigh Boats
80% Ethanol in Spray Bottle (2) Stirring Rods
1 Gallon Distilled Water 5 cm Stirring Magnet
(1) 100 mL Beaker Electronic Balance (0.0001 g accuracy)
(2) 250 mL Beakers Hot Plate
(1) 500 mL Beaker Microwave
(2) 1.0 L Beaker 37C Incubator
250 mL Erlenmeyer Flask Refrigerator
25 mL Graduated Cylinder Fume hood
100 mL Graduated Cylinder Aluminum Foil
(24) 100 mm x 15 mm Petri Dishes Whatman Filter Paper No. 1
(5) Test Tubes Hot Mitt
1000 L Pipette (0.01 mL) Gloves
Pipette Tips Goggles
Inoculating Loop Tongs
Bunsen Burner Graphing Paper
Striker Ti-Nspire Calculator

Procedures:

Silver Nitrate Preparation:

1. Follow standard operating procedures by sterilizing all materials and the lab area
using 80% ethanol, tongs, and boiling tap water in a 250 mL beaker on a hot
plate.

2. Using a weight boat, measure 0.17 g of the solid silver nitrate, AgNO3 and place it
into a 1.0 L beaker.

3. Label the beaker Silver Nitrate solution and place it under the fume hood.

Leaf Extract Preparation:

1. Using the analytic balance and a weigh boat, measure 16.0 g of Camellia sinensis
using a scoopula.

2. Measure 160 mL of distilled water using a 250 mL beaker.

 Marougail-Pushani 16

3. Place the 16.0 g of the leaf extract into the 160 mL of distilled water.

4. Boil the solution for 2 minutes on a hot place and mix will with a stirring rod.

5. After the solution cools, using the Whatman filter paper No. 1, Bchner funnel,
and vacuum suction, filter the solution into a 250 mL Erlenmeyer flask.

6. Label the beaker Leaf Extract solution and store at 4C for 24 hours for further
use.

Green Synthesis AgNPs Preparation:

1. Measure out 160 mL of the 1 mM silver nitrate solution and pour it into a 250 mL
beaker.

2. Measure out 12 mL of the stored leaf extract solution using a 25 mL graduated

cylinder.

3. Pour the 12 mL of the leaf extract solution into the 250 mL beaker of the 1mM
silver nitrate solution.

4. Label the beaker Green AgNPs, wrap it in aluminum foil, and place it under the
fume hood.

Sodium Citrate Preparation:

1. Using the analytic balance, measure 1.4705 g of the sodium citrate into a weigh
boat using a scoopula.

2. Measure 0.5 L of distilled water into a 500 mL beaker and mix the measured
sodium citrate into the water.

3. Place the beaker onto a hot plate and heat it until it dissolves completely.

4. Label the beaker Sodium Citrate solution.

Chemical Synthesis AgNPs Preparation:

1. Retrieve an Erlenmeyer flask, a 10 mL graduated cylinder, a magnetic stir bar, a

stirring rod, and a hot plate and bring them to your lab station.

2. Carefully place the magnetic stir bar in the flask. Measure out 160 mL of silver
nitrate solution, AgNO3 into your Erlenmeyer flask. *Silver nitrate will stain your
skin. Wear gloves and clean all spills immediately.
 Marougail-Pushani 17

3. Heat the silver nitrate solution on the hot plate on medium heat. The magnetic
stirrer will mix the solution while it heats.

4. Insert the pipette tip into the 1000 L pipette and use it to obtain the sodium
citrate.

5. Place the sodium citrate onto a hot plate and once the silver nitrate is at a rolling
boil, slowly add the 6 mL of sodium citrate solution, dropwise, or until the
solution changes color.

6. Five minutes after adding the sodium citrate, carefully remove the beaker from
the heat source with an oven-safe mitt and wait for the solution to cool.

7. Once your solution is cooled, transfer the contents to your beaker. Label the
beaker as Chemical AgNPs.

8. Wrap the beaker in aluminum foil to protect the solution from reacting with light
and place the beaker under the fume hood.

Agar Preparation:

1. Add 1 liter of water to a 1-liter beaker.

2. Place it on a stirring hotplate, set the hotplate on high level.

3. Add a stirring magnet to the 1 liter of water, and place the magnet setting on #4.

4. Slowly add 23 grams per liter of agar powder to the flask. Make sure not to get
the powder onto the sides.

5. Allow the solution to change from a cloudy to a clear liquid, at a temperature of

100C.

6. Remove the solution from the heat and allow it to cool, to be able to pour it out.

7. Adjust the amount of agar to fit needs.

8. Follow the preparation directions located on the agar container.

Plate Pouring:

1. Do not open the cover of the Petri dish completely, to prevent the agar from being
exposed to air, when pouring it into the Petri dish.

2. Always open the lid of the Petri dish without placing the lid on any surface.
 Marougail-Pushani 18

3. Pour the agar so that the bottom of the Petri dish is covered with a thin layer of
agar, about 1-2 mm thick. Pour the agar into the corresponding Petri dishes. Pour
agar into the (+) and (-) controls.

4. Allow the agar to cool, without moving the Petri dish, and then pour agar and 2
mL of the corresponding silver nanoparticles solutions: 2 for the green
synthesized and 2 for the chemically synthesized NPs, as well as 1 for each
control of the silver nanoparticles, making sure to randomize the trials using the
Ti-Nspire calculator (Appendix A).

5. Pour a layer (about 5 mL) of the corresponding AgNPs on top of the agar in the
Petri dish.

Pouring E. coli onto Plates:

1. Obtain the Escherichia coli bacteria slant, transfer loop, testing tube, 1mL pipette,
and the Bunsen Burner. Sterilize the transfer loop and obtain a sample of bacteria
from the slant.

2. Pour 1 mL of distilled water with the pipette into the testing tube and insert the
transfer loop with the bacteria.

3. Stir the transfer loop with the bacteria in the testing tube.

4. Pour the contents into the Petri dish after the agar has cooled.

5. Agitate the Petri dish while rotating it every 10 seconds.

6. Carefully pour out any excess liquid.

7. This method will be used for two Petri dishes of the green synthesis AgNPs and
two Petri dishes of the chemical synthesis AgNPs each day for a total of 8 Petri
dishes for each method. The E. coli cultured each new day will be tested.

Incubation of Dishes:

1. Place the Petri dishes upside down in an incubator set at 37C.

2. Incubate the Petri dishes for 24 hours.

Measuring Percent Growth:

1. Place the circular grid paper on top of the Petri dish and estimate the amount of
bacteria in each square. Find the sum and calculate the percentage of growth.
 Marougail-Pushani 19

Diagram:

Figure 9. How to Measure the Bacteria of a Petri Dish

Figure 9 displays the method of using grid paper to determine the amount of

growth of bacteria grown onto the Petri dish. Also see Data and Observations.

Figure 10. Materials for the Solutions

Figure 10 displays the materials used to prepare the solutions in the experiment.
 Marougail-Pushani 20

Figure 11. Materials for Petri Dish Preparation and Measuring

Figure 11 displays the materials used to prepare the Petri dishes as well as count

the percentage of bacterial growth.

 Marougail-Pushani 21

Data and Observations

Table 1
Observations of Starter Plate, Solution, and Petri Dish Preparation
Observations
Day 1 Two new starter plates of the bacteria were prepared with no complications.
The E. coli in our starter plates grew normally, however we decided to use the
Day 2 first one because it appeared more uniform and normal. The solutions were
prepared successfully.
Day 3 Preparing the Petri dishes: The green synthesis solution appeared slightly foggy.

Table 1 above displays the observation that were recorded of the preparation that

was completed before beginning the trials. This includes observations of preparing the

starter plate on Day 1, creating the silver nanoparticle solutions on Day 2, and preparing

the first set of Petri dishes with their treatments on Day 3.

= 75
= 25

= 100
75
25
= 75 100
= 33.33%

Figure 12. Sample Calculations

Figure 12 above is a sample calculation that displays how the percentage growth

of bacteria was calculated. Grid paper was placed on top of a Petri dish, and the areas that

showed growth of E. coli were shaded. Next, the shaded boxes were counted in order to

determine the number of boxes that contained bacterial growth. The percentage of

bacterial growth was calculated by dividing the number of boxes counted that showed E.

coli growth on the Petri dish by the total area of the Petri dish, which is equal to 75

boxes.
 Marougail-Pushani 22

Shaded boxes
represent E. coli
growth.

Non-shaded boxes
represent no E. coli
growth.

Figure 13. Measuring Percentage Growth

Figure 13 above shows how percentage growth was measured. This process is

described in the sample calculations in Figure 12.

Table 2
Description of Treatment Types
Type Description
The Petri dishes treated with the green synthesized silver
Green Synthesis
nanoparticle solution using green tea leaf extract (bacteria).
The Petri dishes treated with the chemically synthesized silver
Chemical Synthesis
nanoparticle solution using sodium citrate (bacteria).
(+) Control The Petri dishes with agar and E. coli.
(-) Control The Petri dishes with only agar.
The Petri dishes with only agar and the green synthesized silver
Green Control
nanoparticle solution using green tea leaf extract (no bacteria).
The Petri dishes with only agar and the chemically synthesized
Chemical Control
silver nanoparticle solution using sodium citrate (no bacteria).

Table 2 above describes each of the treatment types from which data was

collected from.
 Marougail-Pushani 23

Table 3
Data of First Trials
Boxes of Percentage
Day Type
Growth of Growth
Green Synthesis 6.20 8.27
Green Synthesis 3.00 4.00
Chemical Synthesis 20.0 26.7
Chemical Synthesis 22.0 29.3
1
(+) Control 70.0 93.3
(-) Control 0.00 0.00
Green Control 0.00 0.00
Chemical Control 0.00 0.00

Table 3 above displays the data that was recorded of the first set of trials. The

boxes of growth that were counted when measuring the bacterial growth were recorded.

Additionally, the percentage of growth was calculated by determining the percentage of

boxes counted out of the total number of boxes that made up the area of the Petri dish and

was then recorded.

Table 4
Observations of First Trial
Observations
The Petri dishes that were treated with the green
Green
synthesis solutions had a lower percentage of bacterial
Synthesis
growth compared to the chemical synthesis.
The Petri dishes that were treated with chemical
Chemical
Day 1 synthesis solution had a higher percentage of bacterial
Synthesis
growth compared to the green synthesis.
The positive control showed a significant amount of
Controls bacterial growth. The negative controls had no
bacterial growth.

Table 4 above displays the observations that were recorded of the first trial. The

observations are separated into observations for Petri dishes that were treated with the

green synthesis silver nanoparticle solution, the Petri dishes that were treated with the
 Marougail-Pushani 24

chemical synthesis silver nanoparticle solution, and the Petri dishes that were used as

positive and negative controls. The observations correspond to the data in Table 3.

Table 5
Data of Second Trial
Boxes of Percentage
Day Type
Growth of Growth
Green Synthesis 5.70 7.60
Green Synthesis 2.30 3.07
Chemical Synthesis 18.0 24.0
Chemical Synthesis 17.0 22.7
2
(+) Control 72.5 96.7
(-) Control 0.00 0.00
Green Control 0.00 0.00
Chemical Control 0.00 0.00
Table 5 above contains the data that was recorded of the Petri dishes in the second

trial. Once again, the data includes the number of boxes that were counted that showed E.

coli growth, as well as the percentage of growth.

Table 6
Observations of Second Trial
Observations
The Petri dishes that were treated with the green
Green
synthesis solution had a lower percentage of bacterial
Synthesis
growth.
The Petri dishes that were treated with the chemical
Chemical
synthesis solution had a higher percentage of
Synthesis
Day 2 bacterial growth.
The positive controls showed a significant amount of
bacterial growth, while the negative controls had no
Controls bacterial growth. However, the experiment next to us
had difficulties with their water hose and may have
altered the results of our experiment.

Table 6 contains the observations that were recorded of the second trial. The

observations correspond to the data of the second trial in Table 5.

 Marougail-Pushani 25

Table 7
Data of Third Trial

Boxes of Percentage
Day Type
Growth of Growth
Green Synthesis 2.10 2.80
Green Synthesis 3.20 4.27
Chemical Synthesis 14.5 19.3
Chemical Synthesis 19.3 25.7
3
(+) Control 74.0 98.7
(-) Control 0.00 0.00
Green Control 0.00 0.00
Chemical Control 0.00 0.00
Table 7 displays the data that was collected on the Petri dishes in the third trial.

Table 8
Observations of Third Trial
Observations
The Petri dishes that were treated with the green
Green synthesis solution had a lower percentage of bacterial
Synthesis growth. The silver nanoparticles has a variety of
shapes, when observed under a microscope.
The Petri dishes that were treated with the chemical
Day 3 Chemical synthesis solution had a higher percentage of bacterial
Synthesis growth. The silver nanoparticles were more uniform in
shape, when observed under a microscope.
The positive control showed a significant amount of
Controls bacterial growth. The negative controls had no bacterial
growth.

Table 8 displays the observations of the third trial; these observations correspond

to the data in Table 7.

 Marougail-Pushani 26

Table 9
Data of Fourth Trial

Boxes of Percentage
Day Type
Growth of Growth
Green Synthesis 6.50 8.67
Green Synthesis 4.50 6.00
Chemical Synthesis 18.8 25.1
Chemical Synthesis 15.5 20.7
4
(+) Control 66.0 88.0
(-) Control 0.00 0.00
Green Control 0.00 0.00
Chemical Control 0.00 0.00

Table 9 contains data of the fourth and final trial of the experiment.

Table 10
Observations of Fourth Trial
Observations
Green The Petri dishes had a lower percentage of bacterial
Synthesis growth.
Chemical The Petri dishes had a higher percentage of bacterial
Synthesis growth.
Day 4
The positive control showed a significant amount of
bacterial growth. The positive control grew lass
Controls
bacteria than previous days. The negative controls
had no bacterial growth.

Table 10 displays the observations that were noted during the fourth and final trial

of the experiment, as they correspond to the data of the fourth trial displayed above.
 Marougail-Pushani 27

Figure 14. Chemical Synthesis Silver Nanoparticle Solution and Sodium Citrate

Figure 14 is an image that shows the chemically synthesized silver nanoparticle

solution next to the sodium citrate. The sodium citrate was placed next to the chemically

synthesized silver nanoparticle solution in order to highlight the difference in the color.

As you can see, the silver nanoparticle solution is a very light shade of yellow. This late

shade of yellow indicates small sized nanoparticles. The lighter the shade of yellow, the

smaller the silver nanoparticles are.

Figure 15. Green and Chemical Synthesis Nanoparticle Treatments on Petri Dishes

Figure 15 displays two Petri dishes. The Petri dish to the left is one that was

treated with the green synthesis silver nanoparticle solution, while the Petri dish to the

right is one that was treated with chemical synthesis silver nanoparticle solution. The
 Marougail-Pushani 28

green synthesized silver nanoparticles produced a rosy tint to the Petri dishes as well as

less E. coli growth, whereas the chemically synthesized silver nanoparticles produced a

clearer Petri dish with more E. coli growth.

 Marougail-Pushani 29

Data Analysis and Interpretation

Data was collected of the percentage of Escherichia coli growth in each Petri dish

in order to compare the effects of the green synthesized and chemically synthesized silver

nanoparticles. After the E. coli starter plate was created and the green synthesized and

chemically synthesized silver nanoparticle solutions were created, eight trials were

conducted for each treatment in order to determine which method of silver nanoparticle

synthesis, green or chemical, would be more effective in inhibiting the growth of E. coli.

For each run, two Petri dishes were treated with the green silver nanoparticles and two

Petri dishes were treated with the chemical silver nanoparticles. Additionally, positive,

negative, green synthesis, and chemical synthesis controls were used. During the trials,

each Petri dish was given the appropriate treatment and incubated overnight; the Petri

dishes were observed and data was collected the following day.

The percentage growth of bacteria was calculated through the use of a grid. Grid

paper was placed on top of a Petri dish, and the areas that showed E. coli growth were

shaded in. Next, the shaded boxes were counted in order to determine the number of

boxes that contained bacterial growth. The percentage of bacterial growth was calculated

by dividing the number of boxes counted that showed E. coli growth on the Petri dish by

the total area of the Petri dish, which is equal to 75 boxes. It was hypothesized that the

green treatment would be more effective in inhibiting the growth of the E. coli than the

chemical treatment.

Descriptive analysis was used to analyze the data collected in this experiment.

This method of analysis was the most appropriate for this experiment because there were

only eight trials for each treatment; therefore, there were not enough trials create a
 Marougail-Pushani 30

boxplot, because it would act as a statistically visual error, which would deceive the eye.

Furthermore, running a statistical test was not appropriate for the data proves a clear

significant difference between the two treatments (see below).

The data collected is valid due to the uses of control, randomization, and

replication methods. Four different control types were used: a positive control, a negative

control, a green synthesis control, and a chemical synthesis control. The positive control

was only treated with agar and bacteria; this control serves to show that the bacteria is

growing normally when not treated with any inhibiting solution. The negative control

was only treated with agar; the purpose of this control was to ensure that there was no

contamination in some part of the experiment that would be a lurking variable and affect

the results of the experiment. The green synthesis control was only treated with agar and

the green synthesized silver nanoparticles, while the chemical synthesis control was only

treated with agar and the chemically synthesized silver nanoparticles. These two controls

also made sure that no contamination was evident in the experiment. The randomization

method was applied using the Random Integers option on the TI-Nspire calculator to

randomize the order of the trials for each run. Randomization ensures that the data is

valid by reducing bias associated with having all of the trials performed in the same order

each time. Furthermore, replication was accounted for by doing eight trials for each

treatment type. Replication ensures that the data is valid by reducing variability among

the data. The results and interpretation of the data collected are presented below.
 Marougail-Pushani 31

Figure 16. Dot Plot of Percent of Bacterial Growth

Figure 16 above is a dot plot that displays the data collected of the percentage of

bacterial growth for each treatment. The dot plot displays eight points that represent the

percent of bacterial growth of the eight Petri dishes that were treated with the green

synthesized silver nanoparticles and another eight points which represent the percent of

bacterial growth in the Petri dishes that were treated with the chemically synthesized

silver nanoparticles.

It is noticeable that the two treatment groups are significantly different, as they

are far in range and have no overlap whatsoever. For this reason, a statistical test such as

a two-sample t-test is not appropriate, for it is already clear that the percentage of

bacterial growth is significantly different for each treatment.

 Marougail-Pushani 32

Table 11
One-Variable Statistics of Two Treatments

Table 11 above displays the one-variable statistics for the percentage growth of

bacteria of both the green treatment and the chemical treatment.

As seen above, the mean, or average, of the percent growth of the green treatment

on the E. coli is 5.59%, which is far less than the average of the percent growth of the

chemical treatment on the E. coli, which is 24.2%. The median of the green treatment is

5.14%, which is quite close to the mean. The median of the chemical treatment is 24.5%,

which is also quite close to the mean. This similarity indicates that the distribution of the

data is nearly symmetric and that the actual data is reliable. Additionally, the minimum of

the green treatment is 2.80% and the maximum is 8.67%, for a range of 5.87%. The

minimum of the chemical treatment is 19.3% and the maximum is 29.3% for a range of

10.0%. This shows that there is a wider distribution in the chemical treatment than the

green treatment, which is also indicated visually by the dot plot in Figure 16 above.

Furthermore, the standard deviation of the green treatment (2.37%) is less than the

standard deviation of the chemical treatment (3.25%). The standard deviation simply
 Marougail-Pushani 33

demonstrates how far the samples are from the mean, which provides a good depiction of

the spread of the data. In this case, the spread of the percentage growth is smaller for the

green treatment than for the chemical treatment indicating that the data points are spread

out over a wider range of values, providing more variability for the chemically

synthesized silver nanoparticles.

Overall the descriptive analysis and interpretation of the data clearly displays the

significant difference in the percentage growth of the green treatment versus the chemical

treatment on the bacteria. The data is symmetric and normal for each treatment

supporting reliability, which is also supported by the controls of the experiment, which

ran successfully each day (see Data and Observation). The smaller percentage of growth

of E. coli of the green synthesized silver nanoparticles is in accordance with the

hypothesis (see Conclusion).

 Marougail-Pushani 34

Conclusion

The purpose of this experiment was to determine which method of synthesizing

silver nanoparticles, green synthesis, using Camellia sinensis (green tea) leaf extract as a

reducing agent, or chemical synthesis, using trisodium citrate dihydrate as a reducing

agent, would be more effective in inhibiting the growth of Escherichia coli. A 1.0 mM

solution of silver nitrate, AgNO3 was created and the two different reducing and

stabilizing agents were added into two different beakers to create the silver nanoparticles.

Then two L of each silver nanoparticle solution was added to agar, and the bacteria was

streaked onto the dish and incubated. The percentage growth of the bacteria in each Petri

dish was measured in order to determine which methodology of producing silver

nanoparticles was more effective in preventing microbial growth. It was hypothesized

that the green synthesized silver nanoparticles would be more effective than the

chemically synthesized silver nanoparticles. After using descriptive statistics to analyze

the data, it was revealed that the optimal silver nanoparticle solution that best inhibited

the growth of the E. coli was the silver nitrate reduced by the green tea leaf extract,

meaning that the green silver nanoparticle synthesis was more effective in preventing

the growth of bacteria. An in-depth analysis of the data provided evidence that the results

of this experiment were conclusive.

Silver nanoparticles are known for their antimicrobial capabilities and have been

used to prevent and treat various diseases. These silver ions prevent the growth of

bacteria through a potent defense system by disrupting the replication and respiratory

process of the microbes. The results of this experiment correspond to the scientific

knowledge of silver nanoparticles and their properties. Experimental results demonstrated

 Marougail-Pushani 35

that the green synthesized silver nanoparticles were more effective in inhibiting bacterial

growth.

The scientific understanding is that smaller nanoparticles and more triangular-

shaped nanoparticles are more easily able to bind to the bacterium and ultimately destroy

it. A triangular, sharper shape provides a more efficient way to burst and attack the cell

wall of that certain microbe. The results of this experiment demonstrated that silver

nanoparticles experience a shape and size dependent interaction with the gram-negative

organism, E. coli (Heiden). Furthermore, scientific knowledge explains that silver

nanoparticles that are treated chemically, using sodium citrate are about 30 nm to 60 nm

in size, while those that are created through green synthesis using plants or

polysaccharides, such as Camellia sinensis, are on average 25 nm in size (Siauw,

Winnie). Due to the fact that the green synthesized silver nanoparticles were more

effective in inhibiting the growth of E. coli in this experiment, it can be concluded that

the green synthesized nanoparticles were both smaller in size and more triangular or

sharper in shape.

When sodium citrate is added dropwise to silver nitrate, the excess sodium citrate

present in the solution caps or stabilizes the particles through the carboxylate groups

and prevents them from growing as larger particles, thus acting as both a reducing and

capping agent, and successfully producing silver nanoparticles (Lacson, Gene). However,

chemically synthesized silver nanoparticles are still in their developing stages in which

the process actually experiences problems in its stability and aggregation of

nanoparticles, control of crystal growth, and morphology (Iravani, S., et al). This means

that as time goes on, the silver nanoparticles increase in size and vary in shape, creating a
 Marougail-Pushani 36

large size and shape distribution. This large size and shape distribution in the chemically

synthesized silver nanoparticles makes it more difficult for the nanoparticles to bind to

and attack the cell walls in order to destroy the bacterium. However, green synthesized

silver nanoparticles are extremely stable and do not show signs of disruption or

aggregation meaning they remain the size and shape in which they were originally

created. (Iravani, S., et al). For this reason, the green synthesized silver nanoparticles

remain small in size, which allows them to easily bind, attack, and destroy the bacterium,

thereby more effectively inhibiting the E. coli growth.

The results of this experiment agree with current work in this field. As

aforementioned, current work proves that chemically synthesized silver nanoparticles,

although effective, are still in development stages, for the nanoparticles tend to lack

stability even when using a capping agent. The nanoparticles tend to aggregate and have

a higher size and shape distribution over time. This is why the chemically synthesized

nanoparticles were deemed less effective in inhibiting microbial growth. Furthermore,

current research also states that the green approach to synthesizing silver nanoparticles

is more effective for a number of reasons. The green method stabilizes the nanoparticles

more efficiently and allows for their stability to continue over time. Results have

displayed that the nanoparticles remain un-aggregated for months after their synthesis,

allowing them to have the same shape and size from when they were originally created.

This current research on the green synthesis further supports why the green synthesizes

silver nanoparticles were more effective in inhibiting microbial growth.

Errors in the experimental design negatively affected the data and data analysis.

Errors in the experiment may have resulted from the estimates that were made when
 Marougail-Pushani 37

measuring the percentage growth of E. coli in each Petri dish. Although the data proved

to be significantly different for each of the two methods of synthesis, the numbers

recorded were not entirely exact, but merely a close estimate. Another error in the

experiment was that two different batches of agar were used in a day of trials, meaning

that the bacteria for that day could have grown more or less than the other days of trials,

however, the data displayed no outliers. Additionally, when the silver nanoparticle

solutions were added to the agar, the AgNPs might not have spread across the entire Petri

dish even when the dishes were shifted up and down, left and right, for maximum

coverage; this could have allowed for the bacterial growth in the areas not entirely

covered, as can be seen in Figure 15 of the Data and Observations section of this paper.

Also, due to time constraints, only 8 trials of each synthesis type was run allowing for

only minimal analysis. A lurking variable of the experiment was the incubation

temperature of the Petri dishes. Although the temperature was checked every day to make

sure it was at 37C, it may have changed throughout the day, affecting the growth of the

bacteria. If this experiment was rerun, the experiment would be run in an isolated,

entirely sterile environment. Additionally, advanced equipment would be used to analyze

both the nanoparticles and the bacteria grown. Although errors presented themselves in

the experiment, most errors were controlled due to the control methods used in the

experiment, which proved that no contamination was present and that trials ran

successfully.

Further research in this area can enhance scientific knowledge of silver

nanoparticles and their effects. Silver nanoparticles are known for their antimicrobial

properties; for this reason, further research should be augmented to determine whether
 Marougail-Pushani 38

these silver nanoparticles will prove effective in preventing or treating various specific

conditions, diseases, and infections. For example, an experiment can be conducted to test

the effects of silver nanoparticles on acne. In this experiment, silver nanoparticles may be

used as a treatment for common acne-forming bacteria including propionibacterium

acnes and staphylococcus epidermidis. The effectiveness of the nanoparticles in treating

acne can also be observed by conducting an experiment that compares the effects of other

common acne-treatment types including antibiotics, retinoids, and other topical creams,

to the effects of the silver nanoparticles. Additionally, other metal nanoparticles can be

tested for their antimicrobial properties. Little research involving metal nanoparticles has

been done on people, because certain metals accumulated in the human body may prove

to be highly toxic; for this reason, experimentation has restricted the use of nanoparticles

mostly to topical ointments for skin infections. However, certain metals, like the metal

gallium is safe enough for people to be injected in the human body. Gallium is safe

enough to for humans because it is already present in the human body. Thus, gallium can

be tested for its antibacterial properties on human beings; specifically, the idea that

gallium is a possible intravenous treatment for lung infections can be investigated ("Drug

Development"). Finally, silver nanoparticles were observed and tested in this experiment

as a viable alternative to antibiotics that potentially solves the problem of antibacterial

resistance. Therefore, further research can be done to investigate other alternatives to

antibiotics that would solve the problem of antibacterial resistance. One such potential

alternative that can be researched is bacteriophage therapy. Phage therapy is the

therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.

Bacteriophages or "phage" are viruses that invade bacterial cells and, in the case of lytic
 Marougail-Pushani 39

phages, disrupt bacterial metabolism and cause the bacterium to destruct ("Phage

Therapy"). These bacteriophages can be tested on various bacterial strains, while

observing the bacterial inhibition of each generation of the same bacteria, to determine if

phage therapy is indeed an alternative to antibiotics.

This original experiment was created in order to test the effects of two different

methods of silver nanoparticle synthesis on E. coli in order to determine which was more

effective in inhibiting the bacterial growth. This study will affect future research in the

area because there is a growing need to develop eco-friendly processes, which do not use

toxic chemicals in the synthesis protocols. Nowadays, scientists and manufacturers prefer

to use the chemical synthesis method to create silver nanoparticles because it is less time-

consuming. This research demonstrates that the green synthesis method creates silver

nanoparticles that are more effective in inhibiting the growth of bacteria. This discovery

should lead researchers to use the green synthesis method in order to create nanoparticles

that will more effectively inhibit bacterial growth. Green synthesis approaches include

mixed valence polyoxometalates, polysaccharides, Tollens, biological, and irradiation

methods. These all have advantages over conventional methods involving chemical

agents associated with environmental toxicity. Green synthesis of silver nanoparticles has

advantages over the chemical and physical method as it is money-saving, eco-friendly,

and a one-step method, for the plants do not need separate capping agents as they both

reduce and stabilize the nanoparticles. They are also easily scaled up for large-scale

synthesis and do not require high pressure, energy, temperature, and toxic chemicals for

production, unlike the chemical approach. This research reveals this effective, eco-

friendly, and cheap method to synthesize silver nanoparticles and will revolutionize
 Marougail-Pushani 40

medicine and benefit the human society by allowing for the creation of nanoparticles that

will better prevent and treat human disease, and thereby improve the quality of human

life.
 Marougail-Pushani 41

Acknowledgements

Thanks and appreciation are given to the people that guided and encouraged us

through the course of this experiment. We would like to acknowledge Mrs. Hilliard for

her guidance and support with our creation and execution of this research experiment.

Her supportive, positive reinforcements pushed this experiment to reach its full potential.

We would like to acknowledge Mrs. Tallman for being helpful and supporting this

experiment by perfecting the data and its interpretation. We would like to acknowledge

Mrs. Gravel for helping perfect the format of the paper and always instilling positive and

encouraging thoughts along the way. Finally, we would like to acknowledge our

professional research contact, Patricia Heiden, Ph.D., a professor at Michigan

Technological University, who provided valuable advice and guidance for the creation

and execution of this experiment.

 Marougail-Pushani 42

Appendix A: Randomization

Materials:

Ti-Nspire Calculator

Procedures:

1. Turn on the calculator and open up a calculator page.

2. Press the menu button and scroll to choice 5: Probability and press enter.

3. Scroll to choice 4: Random and press enter.

4. Scroll to choice 6: Seed and press enter.

5. The calculator page will now say RandSeed. Enter any number to seed the

calculator for randomization purposes.

6. Repeat steps 2 and 3.

7. Scroll to choice 2: Integer and press enter.

8. The calculator page will now say randInt(). In the parenthesis, enter one comma

eight meaning the count will start at one and end at eight, in correspondence to the

different trials: chemical AgNPs, green AgNPs, and the controls.

9. Press enter and the number will appear. This number determines which trial will

be run.

10. Keep pressing enter until each trial is assigned to a given trial number.
 Marougail-Pushani 43

Appendix B: Solution Preparation Calculations

Calculations of Silver Nitrate Amount:

=

= 0.001 169.87 1
= 0.16987
Figure 17. Silver Nitrate Solution Calculations

The figure above displays the calculations for creating the silver nitrate solution in

the experiment. This means that 0.16987 grams of silver nitrate per 1.0 liter of distilled

water is needed to create a 1mM solution.

Calculations of Sodium Citrate Amount:

=

= 0.01 294.10 0.5
= 1.4705
Figure 18. Silver Nitrate Solution Calculations

The figure above displays the calculations for creating the silver nitrate solution in

the experiment. This means that 1.4705 grams of trisodium citrate dihydrate is needed per

1.0 liter of distilled water is needed to create a 0.01 M solution.

 Marougail-Pushani 44

Appendix C: Profession Consultant Contact Form

Senior Research Profession Consultant Contact Form:

Name: Veronica Marougail and Frosilda Pushani

Research Topic: Determining Which Method of Synthesizing Silver Nanoparticles is

Most Effective in Inhibiting the Growth of Escherichia coli

Professional Contact Information:

Name: Patricia Heiden, Ph.D.

Title: Professor

Organization: Department of Chemistry at Michigan Technological University

Phone (area code and extension): (906)-487-3452

Email: paheiden@mtu.edu

Mailing Address: Michigan Technological University,

1400 Townsend Drive,
Houghton, Michigan 49931
Dialogue Information:

1) What are some improvements we can make to our experiment?

2) How do we prepare the green synthesis silver nanoparticles?

3) How should we measure bacterial growth?

Additional Information:

Heiden assisted in the green synthesis of the silver nanoparticles, providing various

ways to create them. She also helped in the understanding of other factors that contribute to

the effectiveness of nanoparticles such as size and shape.

 Marougail-Pushani 45

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