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Biotox Teacher Manual 2014 (1)

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Silver Nanoparticle Biotoxicity Experiment
The California NanoSystems Institute
University of California, Los Angeles
High School Nanoscience Institute
Mike Thompson, Laura Schelhas, Ph.D., Kayla Roeser, Helena Chia, Theresa Nguyen, Kristofer Marsh,
Robert Boutelle, Richard Sportsman, Jeff McCormick, Grace Huang
March 2014
INTRODUCTION
This workshop is designed to demonstrate the biotoxic nature of silver nanoparticles (particles with
dimensions less than 1 x 10-7 meter) to microbes. This is a hands-on activity during which participants
synthesize silver nanoparticles and compare their inhibition of yeast respiration (release of carbon dioxide
as a result of their conversion of sugar to energy) to that of silver powder and silver ions in solution. By
adding sugar to yeast in warm water, the yeast cells will increase their respiration rate producing
significant carbon dioxide and water. Pressure changes recorded by a student-made manometer will be
used to measure the amount of carbon dioxide generated in this experiment. The degree of inhibition of
respiration by various silver preparations will be tested, demonstrating the high relative toxicity of a
nanoparticle solution (depicted below). This multidisciplinary workshop engages teachers and students in
discussions about yeast, nanotechnology, current advances in understanding biotoxicity of
nanoscale materials, and the chemistry of nanoparticle synthesis.
For the latest update to the manuals, visit
http://cnsi.ctrl.ucla.edu/nanoscience/pages/homepage
or email: [email protected]
Copyright © 2009 by Sarah Tolbert
Revised 2014
UCLA-CNSI
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Common Core Standards
In order to connect our experiment to the Common Core Standards, we suggest that teachers emphasize
the following lab components, discussion topics, and written excercises. These notes specifically outline
which parts of the lab exercise are intended to help the students generate hypotheses, visualize concepts,
analyze data, and examine scientific literature.
1. Make student thinking visible
Prelab questions:
 Draw a picture of a cell membrane that illustrates different ways in which molecules and
small objects can enter a cell.
 Relate the surface area:volume ratio to radius for spheres of various sizes. Which
nanoparticles have a higher SA/V, large ones or small ones?
 Create an experimental flowchart diagramming the procedure for the lab.
2. Provide opportunities for discourse





Students should discuss the answers to their prelab before working on the experiment
together.
Students should discuss within their group and formulate a hypothesis about which form of
silver will kill the yeast fastest. They should document their hypothesis, and also state what
line of reasoning led them to their hypothesis.
This experiment provides a good opportunity to review the concept of scale in the physical
world. Our understanding of nature, and even just of our simple experiment, spans many
orders of magnitude. (i.e. Ag+ ion = 10-10m; nanoparticle = 10-8m; yeast cell, silver powder
= 1-3 x 10-6m; diameter of milky way galaxy=1021m)
Students should discuss why is is possible to use the manometer to measure the respiration
of yeast, emphasizing that respiration releases gas into the manometer, leading to an
increase in pressure that pushes the water up the tube.
We provide a list of additional discussion questions in Appendix C.
3. Lab Report Elements
Prelab Assignment:
 See section above on “making student thinking visible.”
 The prelab is designed to highlight biological and physical concepts that are key to
understanding the results of the experiment, and also to familiarize students with the lab
procedure to help make the experiment run more smoothly.
 The students should read the background information and the lab procedure, and then
complete the prelab assignment before performing the experiment.
Lab Notes:
Copyright © 2009 by Sarah Tolbert
Revised 2014
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
Students will have a worksheet to complete as they perform the experiment on Day 1.
They will be required to record their hypotheses, as well as their observations and data.
Lab Report:
 The lab report is intended to guide students scientific reasoning as they quantitatively
analyze their results on Day 2.
 Students will evaluate the quality of their experiments.
 Students will learn the science behind why nanoparticles are effective antimicrobials.
 Students will analyze the hypothetical results of an imaginary experiment to further
explore the antimicrobial effect of silver nanoparticles.
 Students will review a news article highlighting research into copper as an antimicrobial
surface. They will be asked to formulate scientific ideas based on the text.
4. Data Analysis and plots
This experiment provides multiple opportunities for students to perform quantitative data analysis:
 In the prelab, students are asked to evaluate the relationships between geometric properties
of spheres.
 During the experiment, students will make quantitative measurements of gas evolution
with respect to time. These results will be plotted, and approximate metabolic rates can be
calculated by finding the slopes of the best fit lines.
 In the lab report, students will plot data and perform quantitative analysis of imaginary
data for another related experiment.
Outline of the Class Activity
Day One
Teacher Pre-Lab – 30 minutes
Begin warming water
Prepare stock solutions
Student Procedure – 50 minutes
Fabrication of nanoparticles and measurement of carbon dioxide output
Teacher Post-Lab – 5 minutes
Collect Supplies
Day Two
Plot and analyze results, complete lab report
***** Tip for Teachers *****
Read the entire teachers manual before you begin this experiment with your students! There are a
number of ways in which students may be assessed on this experiment. You may choose to assign
some (or all) of the prelab and postlab worksheets, you may ask the students to keep a lab
notebook, or you may ask the students to prepare a different type of lab report following your own
design.
**********
Copyright © 2009 by Sarah Tolbert
Revised 2014
UCLA-CNSI
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BACKGROUND
Silver Nanoparticles
Before the 20th century, the only known method of processing silver was by grinding down silver
metal and reducing it to fine powder. This presented a problem for applications where liquid suspensions
of silver particles were required, since fine silver powder does not stay suspended in solution for long
before settling to the bottom of the liquid. During the 20th century, scientists discovered how to create
silver nanoparticles (Figure 1). Nanoparticles are particles of any material that are less than 100 nm in
diameter. In nanoparticulate form, these tiny metallic crystals can be suspended in a liquid base, which
allows for solution processing.
a
b
2 nm
FIGURE 1. Silver nanoparticle synthesis. a. Silver nitrate provides Ag+ ions that are reduced by sodium
citrate, which itself becomes oxidized. Excess sodium citrate present in the solution caps the particles
through the carboxylate groups and prevents them from growing and aggregating as larger particles; this
procedure is carried out at elevated temperature to decompose the citrate and increase the speed of particle
growth. The ratio of sodium citrate to silver ions causes a change in nanoparticle size. b. Transmission
electron micrograph of the silver nanoparticles and a higher magnification image showing the crystalline
planes, indicating that these are nanocrystals of silver.
Copyright © 2009 by Sarah Tolbert
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On the nanoscale, the properties of a material often change from what is observed for
conventional, bulk materials. One easily observable example is the color. Bulk silver is the typical shiny
silver color we are accustomed to, but silver nanoparticles can be a variety of colors, depending on the
shape and size. Particles that are approximately spherical, and below 20 nm in diameter (such as the ones
that will be prepared in this lab exercise), will display a clear yellow or amber color because they absorb
light very strongly. In fact, a very dilute solution can display a bright color. This is due to a phenomenon
called surface plasmon resonance. When the frequency of an incident light wave matches the oscillation
frequency of the surface electrons on the nanoparticle, that light will be absorbed by the nanoparticle
solution. The oscillation frequency of the surface electrons in the silver nanoparticles is such that the
solutions absorb blue light. The oscillation frequency of the surface electrons changes with particle size,
making smaller particles appear more of a pale yellow color, and larger particles more of an amber, or
brownish color.
Another advantage of nanoscale materials is their massive surface area : volume (or surface area :
mass) ratio. This point is illustrated well by considering the example of silver, which we use in our
experiment. A U.S. silver dollar contains 26.96 grams of coin silver, has a diameter of about 40 mm, and
has a total surface area of approximately 27.70 square centimeters. If the same amount of coin silver were
divided into particles 1 nanometer in diameter – the total surface area of those particles would be 11,400
square meters, which is 4.115 million times greater than the surface area of the silver dollar! This concept
of increasing surface area : volume with decreasing particle size is illustrated in Figure 2.
FIGURE 2. Increase in surface area with decreasing particle size.
Yeast – Saccharomyces cerevisiae
Saccharomyces cerevisiae, the common baker’s or brewer’s yeast, is the organism we will use for
our experiment (Figure 3).
FIGURE 3. DIC micrograph of S. cerevisiae. Cells are approximately 2µm in diameter.
Yeast is a unicellular eukaryotic organism, and therefore it has mitochondria and performs cellular
respiration. It is arguably the most basic of the eukaryotic organisms, and it doesn’t do much but perform
Copyright © 2009 by Sarah Tolbert
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cellular respiration and divide. Its simplicity, however, makes it a favorite organism among biologists.
Often when one wants to study a complex biological system, it is easiest to start with a simple model
organism. Additionally, S. cerevisiae serves as a model organism for the infectious fungus Candida
albicans (the causative agent of oral and genital yeast infections). The biological process we will monitor
in our experiment is cellular respiration. Respiration is the process by which a cell converts glucose (or
other sugars) and oxygen into cellular energy (ATP), carbon dioxide and water.
C6H12O6 + 6O2  6CO2 + 6H2O + energy (ATP)
In eukaryotes, such as yeast and humans, this process occurs in a membrane bound organelles called
mitochondria. The integrity of the mitochondrial membrane, and the proteins embedded within the
mitochondrial membrane, are critical for ATP generation by cellular respiration. We will monitor the
respiratory process by indirectly measuring the amount of carbon dioxide gas released by the yeast
cultures. The efficiency of cellular respiration will be used as an overall indicator of the health of the
yeast.
Antimicrobial Properties of Silver
It has been known for centuries that silver is an effective antimicrobial. Ancient civilizations, such
as the romans, stored fresh water in silver flagons to prevent growth of microorganisms. As early as the
1700s, silver nitrate was used to treat open wounds, and it is still used today to prevent eye infections in
newborns. Additionally, in the early 1900s, the U.S. silver dollar was placed in milk jugs to prevent
contamination.
Modern scientists have discovered that the silver ion, and silver-based compounds, are highly
toxic to microorganisms, showing strong biocidal effects against at least 16 species of microorganism,
including Escherichia coli, and various fungi. The mechanism of silver toxicity relies on the chemistry of
the silver ion (Ag+). Once inside the cell, silver ions catalyze the formation of “reactive oxygen species,”
ROS for short, which are free-radicals generated from oxygen that run amuck inside cells and cause all
sorts of damage to various important molecules. ROS can damage DNA, and an even bigger problem is
that they cause lipid peroxidation. Lipid peroxidation is a chain reaction that results in polar chemical
groups being attached to the nonpolar tails of lipids in a membrane (Figure 4), which creates networks of
linked, immovable lipids. These effects disrupt the hydrophobic interactions that keep the membrane
intact, and ultimately lead to cracking and breaking of the cell membrane. In fact, it is lipid peroxidation
that leads to the hardening and drying of oil-based paint. You certainly wouldn’t want your cell
membranes to dry up like paint!
Along with the cellular membrane, the mitochondrial membranes are also disrupted which obliterates the
mitochondrial proton gradient and the membrane-embedded electron transport chain, both of which are
required for energy (ATP) yielding reactions of oxidative phosphorylation during respiration.
Furthermore, the Ag+ ion can damage key proteins by forming strong interactions with the thiol
(sulfhydryl) R-group of the amino acid cysteine, leading to protein dysfunction.
FIGURE 4 – Lipid peroxidation catalyzed by ROS. Leads to disruption of lipid bilayer.
Copyright © 2009 by Sarah Tolbert
Revised 2014
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PRELAB ASSIGNMENT (FOR STUDENTS)
1.) Draw a picture of a cell membrane, depicting several ways that molecules and other small objects
can enter into a cell.
Answers can include ideas about integral membrane proteins (transporters), diffusion directly
through the membrane (hydrophobic molecules only), or endocytosis.
2.) The surface area (S) and volume (V) of a sphere are related to its radius (r) according to the
following equations:
4
𝑆 = 4𝜋𝑟 2 ; 𝑉 = 3 𝜋𝑟 3
Fill in the table for spheres of different radii (r=1, 2, 3)
Radius
1
2
3
Surface Area (S)
12.6
50.3
113.1
Volume (V)
4.2
33.5
113.1
S/V
3
1.5
1
In the space below, make a graph of radius (x-axis) versus S/V (y-axis).
Graph should include the points in the table above, slope should be negative.
Based on your graph, what can you say about the relationship of a sphere’s radius to its
Surface:Volume ratio (S/V).
As r increases, surface:volume decreases.
Copyright © 2009 by Sarah Tolbert
Revised 2014
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3.) Read the “Experimental Procedure” section below. Prepare a flow chart describing the steps you
will take to complete the lab exercise:
Most answers are acceptable. The goal of this question is to make the students read over the
procedure before doing the lab.
EXPERIMENT: DAY 1 – DATA COLLECTION (TEACHER NOTES)
SUPPLIES PROVIDED BY TEACHER:
- Stir/hot plates (one per nanoparticle synthesis experiment if the hot plate only fits one beaker, otherwise
you can place multiple beakers for multiple experiments on one single plate)
- 1 gallon Distilled water or D.I. water (if not available in your lab you can purchase in most stores)
- 1 gallon container for waste (you can use a plastic or glass container as long as it can hold the waste, for
example, you may use an empty milk jug)
- 400 mL beakers for water baths (one per nanoparticle experiment, you may use any glass container that
can hold the 125 mL flask in place, the smallest in this case is a 400 mL beaker)
- 1 L flask/beaker to heat water for experiments (or a combination of smaller flasks/beakers that combined
add up to a liter of water)
- 200 mL beaker or Erlenmeyer for silver nitrate stock solution
- 400 mL beaker or Erlenmeyer for sodium citrate stock solution
- Balance to weigh out sugar and yeast
- Tape
- Spatula or scoopula
- Stirring rods
- Weighing paper (you can cut standard printing paper or notebook paper into squares)
SUPPLIES INCLUDED IN THE KIT:
Reusable:
6 - Thermometers
15 - 125 mL Erlenmeyer flask (note ** use same flask for nanoparticles synthesis each time as the flask
may stain with silver and should used only for synthesis of silver nanoparticles).
10 - manometer setup supplies (tubing/stopper/pipette tip)
5 - stir bars
Consumables:
3 – vial containing 260 mg silver nitrate (http://www.alfa.com/content/msds/USA/11414.pdf)
Copyright © 2009 by Sarah Tolbert
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15 – vials containing 20 mg silver powder (http://www.alfa.com/content/msds/USA/41599.pdf)
3 – vials containing 1.05 g sodium citrate (http://www.alfa.com/content/msds/USA/A12274.pdf)
3 – 100g ziploc bags sugar
1 – bag of 60g of yeast
1 – box gloves
10 – legal size manila folders
GROUP DISCUSSION
**With your group members, discuss your prelab assignment. During your discussion, develop a
hypothesis about which types of silver will be the most effective at inhibiting cellular
respiration. Record your hypothesis in the space below, and explain the thought process that led
you to this hypothesis:
Any hypothesis is acceptable, provided the students provide a logical explanation for their idea.
EXPERIMENTAL PROCEDURE
Safety
Although the bioactive properties of silver ions have been known for centuries, those of silver
nanoparticles have been studied only recently. Studies are still being conducted on the safety of silver for
a variety of health concerns, as silver nanoparticles are now available commercially in many forms.
Generally, large amounts of silver are required for negative effects to develop in humans. Upon
overconsumption of large amounts of silver, and a condition known as argyria, where the skin is turned a
gray-blue tinge, may develop. Due to the wide use and availability of nanoscale silver, there is also
concern over the long-term environmental impacts. Berkeley has recently become the first city to regulate
nanotechnology safety.
Silver nanoparticles stain your clothing and skin so be careful when working with them. Also,
hot water will be used throughout the experiment. Prevent direct contact. Yeast and sugar can be disposed
of by pouring down the drain. However, experiments containing silver in any form need to be disposed
of via EH&S protocols. Please add table salt (sodium chloride) to precipitate the particles and silver
ions and filter out the solid yeast and silver. This can be thrown in the trash and the filtrate will be
nearly free of silver, which can be poured down the sink. If filtering is not possible in your classroom,
feel free to bring your waste to the next CNSI Workshop.
Teacher Pre-Lab
1. Prepare silver nitrate and sodium citrate stock solutions before class begins:
a. Silver Nitrate solution: add 260 mg silver nitrate provided in kit in labeled vial to a flask
or beaker with 325 mL D.I. water. Cover the solution with aluminum foil if you want to
use it the next day, otherwise you must use the entire solution for that class period because
the solution is light sensitive. Below is the calculation of the silver nitrate solution
concentration:
Copyright © 2009 by Sarah Tolbert
Revised 2014
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b. Sodium Citrate Solution: add 1.05 g of sodium citrate provided in kit in labeled vial to a
flask or beaker with 175 mL D.I. water. Below is the calculation of the sodium citrate
solution concentration:
2.
Prepare the water bath for the nanoparticle synthesis. You will need one water bath for each
nanoparticle synthesis. You will need a beaker that holds a minimum of 400 mL of water to be
able to insert the 125 mL Erlenmeyer flask in the beaker. Fill the beaker no more than half way to
avoid water spilling out, you may use tap water. Place the beaker on a hot/stir plate and insert a
thermometer to monitor the temperature to reach a minimum of 80 ºC.
3. You will need to heat at least 1 L of D.I. water in a flask/beaker to 50 ºC using a thermometer.
This water will be used to run the respiration experiments. This is enough water to run at least 5
control experiments, 5 silver nitrate experiments, and 5 silver powder experiments.
4. It’s up the teacher to decide how many groups the class will be divided into, and also decide how
many experiments can be successfully completed during the time allowed. The kit includes
enough material for students to divide into 10 groups, where each group can do a maximum of 2
respiration experiments. You may have different groups running different experiments in order to
complete all of them at least once in 1 class period. The student manual is designed to have group
A run silver nanoparticle and the control experiment in 1 class period, and group B run the silver
nitrate and silver powder experiments in 1 class period. It is up to you to decide how you want
break it down into 1 or 2 days of experiments.
5. Once all of the experiments are completed, there will be enough data for you to do statistical
analysis. It will be interesting to see the variation among the different groups.
Synthesis of Silver Nanoparticles
1. Make sure the water bath reads at least 80 ºC, and keep it as close to 80 ºC as possible. Your
teacher will have a hot water bath set up prior to your class.
2. Label a dry 125 mL Erlenmeyer flask as B. Be sure the stopper makes a tight seal on this flask
and remove.
3. Rinse the Erlenmeyer with D.I. water before using to remove any dust or dirt. Combine 25 mL of
the silver nitrate solution with 25 mL of the sodium citrate solution in flask B and begin heating in
the water bath while stirring using a magnetic stir bar. Your teacher will have these stock solutions
ready.
4. Continuously monitor this reaction by watching for a color change from clear to a light yellow
(15-20 minutes), refer to Figure 5. If after 20 minutes there is no color change, continue to heat
the solution until you do see a color change to a light yellow. Once the solution begins to turn light
yellow, the silver nanoparticles have formed. Quickly remove from water bath and set aside to
stop the reaction. CAUTION HOT!
Note: The biotoxic activity of silver nanoparticles is related to their size, with the smaller particles
having higher activities when normalized for silver mass content. For example, it is known that
smaller silver nanoparticles are more effective in killing E. coli bacteria than large ones, and
typical diameters of less than 10 nm are the best in this regard. In this experiment, silver
nanoparticles are stabilized from aggregation by adsorption of surface capping groups in the form
of citrate (Figure 1). Once silver nanoparticles aggregate, significant loss of their antibacterial
activity occurs due to the loss of surface area and inability of the large particles to enter the cell by
endocytosis. The synthetic method we use has a larger than 1:1 molar ratio of sodium citrate to
silver, which is necessary for growth and stabilization. A portion of the citrate is used to reduce
the silver ions to silver metal, while the remaining citrate caps and protects the particles from
aggregation. **Important** The size of the silver nanoparticles produced is very sensitive to the
temperature and reaction time. Expect variations in size for each synthesis. The smallest
Copyright © 2009 by Sarah Tolbert
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nanoparticles are a clear yellow color, while increases in size lead to amber, brown, and finally
a cloudy brownish/green color (See Figure 5), which are increasingly less effective as
antimicrobial agents. Note the color of the particles before addition to the yeast and watch for
trends in biotoxicity.
FIGURE 5. Representative nanoparticle syntheses. Remove reactions from water bath while pale
yellow (left); as the particles cool, they will continue to react and turn yellow (middle). If reaction occurs
too long, particles will continue to grow and become cloudy/brown (right) and these are less effective as
antimicrobials.
**After you have completed your nanoparticle synthesis, please record the color of the solution in the
space below. Be descriptive:
Monitoring Yeast Respiration:
Setting Up the Differential Manometer
A differential pressure manometer setup is used to measure the CO2 produced by yeast respiration in
the presence of different forms of silver. The basic measurement setup is illustrated in Figure 6 below.
1. Fold one of the manila folder cover in half.
2. Open the folded cover slightly to form a triangle, with the cover edge leaning against the unfolded
cover.
3. Tape the edge of the folded cover onto the unfolded cover. You now formed a triangular structure
with the manila folder that should be able to robustly stand up on the lab table as shown in Figure
(6).
4. Bend the tubing connected to the stopper and build a manometer by taping the tubing to a manila
folder as shown on Figure (6). Make certain that the tubing is straight to avoid introducing error in
your height measurement.
5. Fill the manometer using a plastic pipet about half full of colored tap water so that level on both
sides is the same, as in Figure (6).
6. This manometer is stable but be careful when running respiration experiments to not let it fall.
You may tape it to the table. Work with your partner.
***Record your data on the data sheets provided***
Copyright © 2009 by Sarah Tolbert
Revised 2014
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tubing
h
Liquid
column
stopper
Flask w/
Yeast
FIGURE 6 – Differential pressure manometer. The schematic on the left represents the differential
pressure manometer that will be used to measure CO2 produced by yeast respiration. Photos on the right
illustrate what the assembled manometer should look like. The water level will change as CO2 is
produced.
Flask A: Control
1. Label a dry 125 mL Erlenmeyer flask as A. Test to make sure the stopper makes a tight seal on
this flask and remove.
2. Weigh out 1.0 g active dry yeast and 2.5 g sugar and add to flask A.
3. Take the tubing connected to the stopper and make a manometer by taping the tubing to a manila
folder as shown on figure (6).
4. Fill the manometer about half full of colored tap water so that level on both sides is the same
5. Add 50 mL warm water (50°C) to flask A, swirl for 10 seconds, wait 30 seconds and plug flask
firmly (so no gas can leak out of flask) with stopper and tubing.
6. Using a pen or pencil, (for experiments B, C and D try to use a different color pen) mark the initial
water height in the manometer on an unmarked area of the manila folder next to the tubing, and on
the data table supplied at the end of the manual. Wait 1-2 minutes until the water starts to move
and then continue to collect data points by making marks every ten seconds (you can change this
time to 20, 30 sec or more according to how fast the water moves) for as long as you can without
water spilling out of the tubing.
7. Discard the contents of flask A down the drain and rinse flask.
Flask B: Silver Nanoparticles
1. Weight out 1.0 g of dry active yeast and 2.5 g of sugar and keep on a dry piece of paper
2. Take flask B from the Silver nanoparticle synthesis above and adjust the temperature to 50 °C by
either heating in the water bath or cooling it with cool tap water
3. Add yeast and sugar to flask B swirl for 10 seconds, wait 30 seconds and plug firmly with stopper
and tubing of the manometer.
4. Using a pen or pencil, mark the initial water height in the manometer on an unmarked area of the
manila folder next to the tubing, and on the data table supplied at the end of the manual. Wait 1-2
minutes until the water starts to move and then continue to collect data points by making marks
every ten seconds (you can change this time to 20, 30 sec or more according to how fast the water
moves) for as long as you can without water spilling out of the tubing.
5. Unplug the stopper before water spills out of the manometer tubing.
Copyright © 2009 by Sarah Tolbert
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6. Collect the contents of flask B in waste container and rinse flask.
7. Using a ruler, record the height of the markings you have just made on the manila folder, using
zero at the initial mark, and transcribe the values into the data table provided at the end of the
manual.
8. Collect the contents of flask D in waste container and rinse flask.
Flask C: Silver Powder
1. Label a dry 125 mL Erlenmeyer flask as C. Test to make sure the stopper makes a tight seal on
this flask and remove.
2. Weigh out 1.0g active dry yeast and 2.5g sugar.
3. Add the yeast, sugar and 20 mg silver powder (provided in small vials, pre-weighed) to flask C.
4. Take the tubing connected to the stopper and make a manometer by taping the tubing to a manila
folder as shown on figure (6)
5. Fill the manometer about half full of colored tap water so that level on both sides is the same
6. Add 50 mL warm water (50°C) to flask C, swirl for 10 seconds, wait 30 seconds and plug flask
firmly with stopper and tubing.
7. Using a pen or pencil, mark the initial water height in the manometer on an unmarked area of the
manila folder next to the tubing, and on the data table supplied at the end of the manual. Wait 1-2
minutes until the water starts to move and then continue to collect data points by making marks
every ten seconds (you can change this time to 20, 30 sec or more according to how fast the water
moves) for as long as you can without water spilling out of the tubing.
8. Collect the contents of flask C in waste container and rinse flask.
Flask D: Silver Nitrate
1. Label a dry 125 mL Erlenmeyer flask as D. Test to make sure the stopper makes a tight seal on
this flask and remove.
2. Add 25 mL warm D.I. water and 25 mL stock silver nitrate solution to flask D and warm to 50°C.
Your teacher will have this stock solution ready for you.
3. Weigh out 1.0g active dry yeast and 2.5g sugar and keep on a piece of paper dry.
4. Take tubing connected to the stopper and make a manometer by taping the tubing to a manila
folder as shown on fig (4).
5. Add yeast and sugar to flask D and swirl for 10 seconds wait 30 seconds and plug flask firmly
with stopper and tubing.
6. Using a pen or pencil, mark the initial water height in the manometer on an unmarked area of the
manila folder next to the tubing, and on the data table supplied at the end of the manual. Wait 1-2
minutes until the water starts to move and then continue to collect data points by making marks
every ten seconds (you can change this time to 20, 30 sec or more according to how fast the water
moves) for as long as you can without water spilling out of the tubing.
7. Collect the contents of flask D in waste container and rinse flask.
Copyright © 2009 by Sarah Tolbert
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Experimental Data Sheet
Control – Flask A – Respiration of Yeast Data
Time Interval
(seconds)
Time To Take
Reading
Change in water
height
Observations
0
10
20
30
40
50
60 (1 min)
70
80
90
100
110
120 (2 min)
130
140
150
160
170
180 (3 min)
Copyright © 2009 by Sarah Tolbert
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Silver Nanoparticles – Flask B – Respiration of Yeast Data
Time Interval
(seconds)
Time To Take
Reading
Change in water
height
Observations
0
10
20
30
40
50
60 (1 min)
70
80
90
100
110
120 (2 min)
130
140
150
160
170
180 (3 min)
Copyright © 2009 by Sarah Tolbert
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Experimental Data Sheet
Silver Powder – Flask C – Respiration of Yeast Data
Time Interval
(seconds)
Time To Take
Reading
Change in water
height
Observations
0
10
20
30
40
50
60 (1 min)
70
80
90
100
110
120 (2 min)
130
140
150
160
170
180 (3 min)
Copyright © 2009 by Sarah Tolbert
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Silver Nitrate – Flask D – Respiration of Yeast Data
Time Interval
(seconds)
Time To Take
Reading
Change in water
height
Observations
0
10
20
30
40
50
60 (1 min)
70
80
90
100
110
120 (2 min)
130
140
150
160
170
180 (3 min)
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EXPERIMENT: DAY2 – LAB REPORT (TEACHER NOTES)
Data Analysis
1. Plot time versus change in height of the column (Δh), you can also plot time vs. number of moles
of carbon dioxide gas if you performed this calculation. This can be done using any commonly
available spreadsheet software, such as Microsoft Excel, more sophisticated data analysis and
graphing software, such as Matlab, or manually on a graph paper. Attach the graphs to your lab
report.
2. Fit the data to a line and notice the slopes of the lines. Determining the slope can be done
manually by dividing rise/run or with graphing software. The larger the slopes, the higher the
respiration rate. If possible, perform a linear regression on the lines to determine if the data is
linear.
3. Compare data to other groups and calculate a mean rate for each of the four experimental
conditions (control, nanoparticles, ions, powder).
Data Analysis Questions
Fill in the table below with the respiration rates calculated for your four yeast cultures, and also fill
in the mean values calculated as a class.
Sample
Control
Nanoparticles
Ions
Powder
Respiration Rate
Class Mean
Based on the results in your table above, evaluate the hypothesis you made before doing the
experiment. Were you correct? Incorrect? If your results did not match your hypothesis, can you
think of another explanation for your data?
Students should provide a critical assessment of the quality of their experiment.
How closely do your data match the mean values calculated as a class? Do you have any reason to
distrust your data? If so why, and what might have caused the discrepancy?
Students should provide a critical assessment of the quality of their experiment.
Copyright © 2009 by Sarah Tolbert
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So Why are Nanoparticles Toxic to Microorganisms?
In the background section, we described the toxicity of the silver ion (Ag+), but the experiment
you just did demonstrates the potent toxicity of silver nanoparticles. Now we must connect the two ideas.
In any crystalline surface, such as the surface of a metal, there is a constant equilibrium involving the
dissociation and reassociation of ions between the metal surface and the solvent. So whenever we have a
metal surface submerged in a solvent, there will be some small amount of ions in solution. The amount of
ions that go into solution depends on the equilibrium constant of dissociation/reassociation, and also on
the amount of surface area that is exposed to the solvent. This is where the high surface area of the
nanoparticles comes into play. A given mass of silver in nanoparticulate form will have much more
surface area than the same mass of bulk silver powder; therefore it will be more active in releasing Ag+
ions to the solution.
In order to have maximum toxicity, silver ions have to get into the cell. This is another aspect
where the unique size of the nanoparticles plays a key role. Typically, ions cannot diffuse into the cell
directly through the plasma membrane because they are charged, and the membrane has a very
hydrophobic interior. Cells do, however, have integral membrane proteins called transporters that are used
to transport some essential ions into the cytoplasm. Silver is not an essential ion, so there is no membrane
transporter that uses Ag+ as its natural substrate, but some silver ions can sneak into the cell slowly
through transporter proteins that are intended to move other similar ions (copper or iron, for example). In
order to get silver ions into the cell, our nanoparticles act as a sort of “Trojan horse.” Nanoparticles that
are 5-20nm in diameter are the right size to be internalized into the cell by endocytosis (Figure 7).
FIGURE 7. Endocytosis.
Once inside the cell, these nanoparticles release silver ions off of their surfaces, which go on to catalyze
all kinds of intracellular mischief as described above. As a consequence, the antimicrobial efficacy of
silver nanoparticles is directly related to their size, with smaller particles being more effective due to their
larger mass-normalized surface area. In contrast to the nanoparticles, bulk silver powder has a small massnormalized surface area, so relatively few ions are released from the surface, and the particles are roughly
the same size as the cells, making it impossible for them to enter the cell by endocytosis or any other
mechanism.
One last question remains. Why are silver nanoparticles seemingly more toxic to yeast than
humans? The short answer is that silver nanoparticles, and silver in general, are toxic to humans.
Ingesting large amounts of silver does have adverse health effects, but in general, humans have more
highly specialized mechanisms for getting nutrients into their cells, and do not rely on endocytosis.
Microbes, because of their relative simplicity, rely on endocytosis to get nutrients past their cell
membranes. Additionally, contact with silver metal (such as silver jewelry and silver coins) is rendered
harmless by the protective outer layers of our skin.
Copyright © 2009 by Sarah Tolbert
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Taking it to the Next Level: An Imaginary Experiment
In the section above, you read about how nanoparticles deliver silver ions to the inside of the cell. This
text mentioned that smaller particles are more toxic, because they have a larger surface area relative to
their mass.
Also, the background material for the experiment described “surface plasmon resonance,” a phenomenon
that causes silver nanoparticle solutions to appear colored. Larger particles absorb more blue light (at
higher energy), and therefore appear a darker shade of red. Knowing that blue light has a wavelength of
about 450nm, and red light has a wavelength of about 700nm. How could you design an experiment to
prove that smaller particles are more toxic to microbes? What would you use as a control?
You could measure the respiration rate of the yeast when exposed to each of the three different solutions.
The control would be the same as for the lab exercise, a culture with no nanoparticles added.
You prepare three nanoparticle solutions. You notice that one solution is pale yellow, another is slightly
orange, and a third is a dark amber color. These three solutions absorb light most strongly at 604nm,
556nm, and 479nm respectively.
Based on your knowledge of how nanoparticles kill microbes, and your knowledge of surface plasmon
resonance, which of your nanoparticle solutions will likely be the most effective at killing yeast cells?
Explain your answer, and make a hypothetical plot of what you might expect your results to look like.
(Plot the maximum absorption of the different solutions on the x-axis, and the values you would expect
for the respiration rates of the cells on the y-axis.)
The particles that are smallest will have their absorbance maximum furthest to the low-energy end of the
spectrum (large wavelength). In the graphs produced by the students, the data points representing the
lowest absorption maximum should have the fastest respiration rate, because the larger particles should
be less toxic.
Copyright © 2009 by Sarah Tolbert
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Application: Using Science to Save Lives
In our experiment, we used nanoparticles to kill common baker’s yeast. While killing these yeast in the
lab demonstrates the antimicrobial properties of silver, ultimately scientists aren’t interested in killing
baker’s yeast. As mentioned before, the yeast serves as a model organism for other pathogenic
microorganisms. Scientists can use the information learned in the lab to develop technologies that are
useful outside of the laboratory. The article on the following page describes the efforts of scientists
who are using copper metal as an antimicrobial in hospitals. Read the article and answer the
following questions:

What is the main problem that these scientists are trying to solve with their research?
Hospital-borne infections are a huge healthcare burden. In hospitals, bacteria persist for
long periods on various surfaces.

In our experiment, we used silver as an antimicrobial. The scientists that you learned about in the
article are using copper instead. Copper and silver have similar antimicrobial properties. Why do
you think this is the case? (Hint: Look at a periodic table.)
Copper in in the same group as silver on the periodic table. Therefore, it has a similar
valence electron configuration and similar chemical properties.

Why do you think copper would be a good alternative to silver for installation in hospitals?
It’s cheaper.

Can you think of another good use for copper (or silver) as an antimicrobial? What about in
nanoparticle form?
Any answer that demonstrates critical thinking is acceptable.

What is the control that these scientists are using in their experiment?
They are comparing their copper surfaces to other surfaces, such as plastic and stainless
steel, which are already used in hospitals.
Copyright © 2009 by Sarah Tolbert
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ARTICLE:
UCLA study to determine if copper surfaces can reduce hospital-acquired infections
$2.5 million grant to fund clinical trial at Ronald Reagan UCLA Medical Center
By Rachel Champeau July 09, 2012
Hospital-acquired infections are a huge public health burden, and hospital environments play a key role in
harboring potentially deadly bacteria such as E. coli, C. difficile and methicillin-resistant Staphylococcus aureus.
These microbes may persist for extended periods in the hospital, on surfaces such as bed rails, doorknobs, chairs,
tray tables, toilet seats and even call buttons in patient rooms.
Copper surfaces, which are not routinely used in hospitals, are known to kill bacteria on contact, and studies have
found much lower levels of bacteria living on copper surfaces than on standard hospital surfaces.
Now, an interdisciplinary team from UCLA is taking this research to the next level. In one of the first randomized
clinical trials of its kind, researchers will determine if the reduction of surface bacteria due to the use of copper will
result in a decreased number of hospital-acquired infections.
Funding for the landmark $2.5 million study will be provided by an RO1 grant (HS021188-01) from the Agency for
Healthcare Research and Quality, part of the U.S. Department of Health and Human Services. The project will
involve teams from the David Geffen School of Medicine at UCLA, the UCLA Fielding School of Public Health
and the Henry Samueli School of Engineering and Applied Science. The collaborative research initiative is a
project of the UCLA Sustainable Technology and Policy Program.
For the clinical trial, two intensive care units at Ronald Reagan UCLA Medical Center will be outfitted with
copper, sham stainless steel, or conventional surfaces such as plastic or other types of coatings. Over a four-year
period, all three surface types will be sampled for bacteria levels, and patient-infection outcomes rates will be
compared among the three surfaces.
"We will be studying if lowering the level of bacteria on hospital surfaces results in reduced infection rates in
patients, better outcomes and even lower costs," said the project's principal investigator, Dr. Daniel Uslan, director
of the antimicrobial stewardship program at the Geffen School of Medicine and an assistant clinical professor of
medicine in the division of infectious diseases.
Additional environmental microbiologic studies and evaluations of surface cleaning will be included in the
research, as well as a detailed cost–benefit analysis.
Dr. Peter Sinsheimer, executive director of the UCLA Sustainable Technology and Policy Program, a joint
initiative of the Fielding School of Public Health and the UCLA School of Law, helped arrange the
interdisciplinary collaborations.
"Being at UCLA makes it easy to pull together diverse teams of top-flight scientists to conduct such important
prevention-based research," said Sinsheimer, whose program focuses on primary health prevention through
materials substitution.
The initial idea for the hospital-based study came from Sinsheimer's research on the viability of alternatives to leadbased copper piping in delivering safer drinking water.
Hospital surfaces selected for the study will include bed rails, chairs, a bedside table that can also be positioned on
top of the bed, and a mobile treatment cart-top used by nursing staff that includes handles, a keyboard and a mouse.
Copyright © 2009 by Sarah Tolbert
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A team at UCLA Engineering will assist with the testing of the copper and other surfaces used in the clinical trial.
"We will be incorporating copper, plastic or sham stainless steel materials into the selected everyday surfaces used
by patients and staff in the hospital," said Vijay Gupta, a professor of mechanical and aerospace engineering.
The cost-effectiveness analysis will be conducted by Dr. Gerald Kominski, director of the UCLA Center for Health
Policy Research and a professor in the department of health policy and management at the Fielding School of
Public Health.
"Finding effective interventions to reduce hospital infection rates in a cost-effective manner is an emerging priority
for U.S. hospitals," Kominski said. "This study will provide valuable information on whether copper-touch surfaces
are a cost-effective technology for achieving this goal."
For more news, visit the UCLA Newsroom and follow us on Twitter.
Article from UCLA newsroom: http://newsroom.ucla.edu/portal/ucla/ucla-receives-2-5-million-grant235213.aspx
Copyright © 2009 by Sarah Tolbert
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APPENDICES FOR TEACHERS
APPENDIX A: SAFETY-RELATED INFORMATION
Here are the links to Material Safety Data Sheets (MSDS) for the three chemicals used in the
Biotoxicity Kit:
Silver nitrate, ACS, 99.9+% (metals basis)
Alfa Aesar - Stock Number 11414
http://www.alfa.com/content/msds/USA/11414.pdf
Silver powder, spherical
Alfa Aesar - Stock Number 41599
http://www.alfa.com/content/msds/USA/41599.pdf
Sodium citrate dihydrate
Alfa Aesar - Stock Number A12274
http://www.alfa.com/content/msds/USA/A12274.pdf
APPENDIX B: ADDITIONAL EXCERCISES
Comparing silver to other established antimicrobials:
In order to illustrate the idea that silver is an effective antimicrobial, it is useful to allow students
to compare the silver solutions to other antimicrobials that they are perhaps more familiar with. If
time and resources allow, an additional experiment can be prepared. This experiment should begin
by preparing 50mL of a dilute solution of a well-known antimicrobial cleaning product (409,
Lysol, hand soap or sanitizer). Warm the solution and add yeast and sugar as described in the
experimental procedure, and then measure the CO2 production with a manometer.
Relating respiration rate to particle size:
If teachers and students have access to a spectrophotometer, it will be possible to measure an
absorption spectrum of the particles prior to adding the yeast. As a class, students can plot
metabolic rates vs. absorbance maxima for their nanoparticle experiments. This will allow a
connection to be drawn between color, an indirect measure of particle size, and efficacy as an
antimicrobial. A similar imaginary experiment is discussed in the student lab report, but a real
experiment can be done if resources exist.
Calculating moles of CO2 produced by cellular respiration using the manometer:
This exercise can be conceptually difficult for students to understand, however it may be wellsuited for advanced classes. The calculations cover the physical basis for quantifying gas evolution
with a manometer, starting from the ideal gas law and using some algebraic calculations.
Completion of this exercise does, however, allow students to calculate a “true” rate of respiration
(in mol/s), as opposed to the “indirect” rate estimation (Δh/Δt) that we recommend for most
classes.
The differential manometer can be explained starting from the ideal gas law:
PV  nRT
Where
P is the pressure of the system
Copyright © 2009 by Sarah Tolbert
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V is the volume of the system
n is the number of moles of gas in the system
R is the gas constant
T is the temperature of the system
At constant temperature, small changes to the system will yield the following equation, where Δ
denotes a small change:
PV  PV  nRT
Or in fractional changes:
n P V


n
P
V

For our setup, we make the following reasonable approximations:
1. The liquid is incompressible (volume does not change with increase pressure).
 expand within the pressure range of our experiment
2. The plastic tubing does not
With these two assumptions, the differential manometer as designed in figure 6 can be further
described with the following statements:
1. The volume change induced by the CO2 production of the yeast is equal the change in the
volume of the gas in the tubing, as reflected by the shift in position of the manometer liquid
column.
V  hA
Where
Dh is the change of height for the liquid column (see Figure 6)
A is the cross sectional area of the tubing (for the tubing provided, A is equal to
2 ml
of water for 5 cm of tubing length.
, i.e. carries 2 ml
5 cm
2. At equilibrium, pressure change in the flask due to the CO2 production must equal the change
of “weight” pressure as the manometer liquid column shift its height against gravity (the
pressure exerted by any liquid column under the force of gravity is proportional to the height
of the liquid column).
P  hg
Where
r is the mass density of liquid column (e.g. density of water)
g is the gravitational constant, equal to 9.8 m/s2

Putting it all together, the fractional change in the number of mole of gas from the
production of CO2 by the yeast is proportional to the change of height for the liquid column
(marked in Figure 6):
n g A 
   h
n  P V 
or
n
 h
n

APPENDIX C: ADDITIONAL DISCUSSION QUESTIONS
Q: What could you do to increase the rate of respiration?
More sugar, higher temperature, more yeast
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Q: Why is it important to keep the water at 50°C?
To ensure the respiration rates and effectiveness of antimicrobials are comparable between
experiments
Q: How does silver interfere with respiration?
Respiration is dependent on the integrity of the mitochondrial membrane. Silver ions catalyze
chemical reactions that damage lipids and destroy these membranes.
Q: What is the most effective microbial agent of those tested in this experiment?
Silver nanoparticles, because the enter the cell by endocytosis and release silver ions locally,
within the cell.
Q: Why does nanoparticle size matter?
Smaller particles enter the cell more effectively via endocytosis, and they have more surface area,
so they release more silver ions once inside the cell.
Q: Why are silver nanoparticles yellow or amber?
Surface plasmon resonance - absorption of blue light by oscillating surface electrons.
Q: What is the molar ratio of sodium citrate to silver nitrate in your nanoparticle synthesis? Why
do we use this ratio?
4.33 : 1 citrate:silver. We need an excess of citrate to both protect the nanoparticles from
aggregation and reduce the silver ions to silver metal. (N.B.: students will need to know the
concentrations of the Na Citrate and AgNO3 solutions to answer this question.)
Q: Assuming the particles are perfect spheres of 4 nm diameter, how many particles are in each 50
mL solution? (density of silver = 10.49 g/cm3)
Volume = 3.35 x 10-20 cm3 (V = 4/3 p r3)
Mass = 3.52 x 10-19 g/particle
20.0 mg silver nitrate = 12.7 mg silver
3.6 x 1016 nanoparticles in 50 mL
Q: Compare the results between the various groups. What is the standard deviation in the
measurements? Discuss what may have been the cause.
Silver nanoparticle effectiveness depends greatly on size and dispersion. The reaction rate of the
particle synthesis can result in changes in size and degree of agglomeration. It is likely that each
group’s nanoparticle synthesis was slightly different, resulting in slight differences in size and
antifungal properties.
Q: How many moles of gas were released per minute? (PV = nRT)
Note: 1 mL =1 cm3, R = 82.0575 atm.cm3/(mol.K), Kelvin (K) = degrees Celsius + 273.15
See Appendix B (additional excercises).
APPENDIX D: ADAPTING THE EXPERIMENT FOR MIDDLE SCHOOL
We feel that this is an excellent experiment for middle school science students as well as high
school students. Middle school students learn about cellular respiration, and are aware that
microorganisms are the causative agents of infection. Additionally, this activity illustrates how chemistry
can be used to address a public health concern, providing a direct example of how science improves our
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everyday lives. Additionally, middle school students learn some of the basic physical/chemical concepts
that appear throughout the lab (such as pressure), and the exercise provides opportunities to discuss those
concepts.
For the middle school audience, we suggest providing students with a more generalized view of
silver as an antimicrobial. For example, it might be useful to exclude the experimental conditions that
include the silver ions (i.e. the AgNO3 solution) to avoid having to get into the details of ions vs.
nanoparticles. It is possible to simply illustrate the point that different forms of silver (i.e. different
particle sizes, nano vs. bulk) have different antimicrobial effects, and we have to use chemistry to access
these useful forms. All of this can be done without having to invoke the concept of surface area and
equilibria, membrane destruction, the details of cellular respiration, or any other advanced topics. It may
be useful, however, to prepare an experiment using a well-established antimicrobial so students can make
a clear connection between the silver nanoparticles, and a substance that they know kills microbes (see
Appendix B).
Finally, when working with a middle school class, it may be helpful to divide the experiment into
more days (i.e. 3 vs. 2). For example, day one could include an introduction to the lab and the students
could do their control experiment. Day two could focus on nanoparticle synthesis and the experiment with
the yeast aand nanoparticles. Day three could include the bulk silver experiment and data analysis. (Note:
nanoparticles must be used immediately after synthesis; do not split particle synthesis and use over two
days.) Alternatively, the labor could be divided between more groups to make things easier for the
students to complete in less time. For example one group could do the control, one the nanoparticle
experiment, and one the bulk silver experiment.
APPENDIX E: ADDITIONAL INFORMATION ABOUT SILVER AS AN ANTIMICROBIAL
More on Historical Uses of Silver as an Antimicrobial:
The earliest record of silver as an antimicrobial comes from Herodotus in 450 BC who told of the
King of Persia keeping boiled water in flagons of silver to keep the water fresh. Ancient Romans would
keep silver pieces in the bottom of milk containers. In the 17th and 18th centuries, silver nitrate was used to
treat open wounds (especially due to burns). It was shown in 1869, that Aspergillus niger could not grow
in silver lined vessels. In 1880, a silver nitrate solution was used to prevent opthalmia neonatorum, a
common eye infection in newborns, and a similar treatment is still used to this day. In the late 1940s,
silver sulphadiazine was used as the standard treatment for burns. It offered general antimicrobial
properties without some of the side effects of antibiotics.
Current Uses of Silver Nanoparticles in Antimicrobials:
Silver and silver nanoparticles are found in a wide variety of products that take advantage of
silver’s antimicrobial properties. Curad and Band-Aid sell bandages coated with silver colloids. In
common medical practice, silver is used in aseptic dressings for plastic surgery, traumatic wounds, leg
ulcers, skin grafts, incisions, abrasions and minor cuts (Aquacel, Acticoat products among others). It is
used to coat catheters and wound bandages. Outside of the medical field, silver is used in institutional
water distribution systems and can be found in Brita water purification systems. Silver is used to sterilize
recycled drinking water aboard the Russian MIR space station and on the NASA space shuttle. In Japan,
silver is mixed into plastics for antimicrobial protection of telephones, calculators, toilet seats, children’s
toys, and pacifiers. Silver can also be found imbedded into athletic garments, sleeping bags, and other
fabrics. See images on next page.
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Current Uses of Silver Nanoparticles in Antimicrobials:
FIGURE A1: Ionic silver starts killing a broad spectrum
of pathogens within 30 minutes of exposure to the
dressing as demonstrated by in vitro testing, including
aerobic and anaerobic bacteria.
FIGURE A2: ACTICOAT Moisture Control is an absorbent antimicrobial
dressing. The product consists on a silver coated wound contact layer, a highly
absorbent foam and a waterproof top film.
FIGURE A3: Silver-nanoparticle-embedded antimicrobial paints on glass slides without coating (a),
glass slides coated with paint only (b) and glass slides coated with silver nanoparticle-embedded paint (c),
onto which E. coli cells were sprayed and incubated at 37 °C overnight. Each black dot corresponds to a
bacterial colony grown from a single surviving bacterial cell. Note lack of growth (dots) on nanoparticleembedded paint (c). (Nature Materials 7, 236 - 241 (2008))
a
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