The Effect of Silver Nanoparticles on the Viability of Bacteria, Fungi, Aquatic Organisms and Plants

Student: Jillian Yao
Table: 1
Experimentation location: Home
Regulated Research (Form 1c): No
Project continuation (Form 7): No

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Abstract:

Many consumer products such as sports clothes, socks, bandages, cosmetics, toothpastes, deodorants, sunscreens and food containers contain tiny silver particles called nanoparticles. Consumer products advertise that these nanoparticles have antibacterial properties that prevent bacteria from growing on everyday items that we use. This project investigated the antibacterial and antifungal effectiveness of different concentrations of silver nanoparticles. Using the Kirby-Bauer testing method to study the effects of nanosilver on Escherichia coli and Saccharomyces cerevisiae, there was a linear relationship between nanosilver concentration and cell death. With increasing concentrations of colloidal silver, there was an increasing diameter of the zone of inhibition. Plating density and incubation times did not have an effect on diameter of the zone of inhibition. At each concentration of colloidal silver tested, S. cerevisiae had larger zones of inhibition versus E. coli suggesting yeast may be more susceptible to colloidal silver. Eight consumer products containing nanosilver particles all showed varying degrees of antibacterial and antifungal activity as measured by the diameter of the zone of inhibition. There was no correlation between the zone of inhibition and nanosilver concentration. However, this could be due to multiple factors including different size, shape, surface area and coating of the silver nanoparticles in these products, and cell death by other reasons such as osmolarity, pH, detergents, and antimicrobial effects of essential oils contained in the products.

In addition to confirming the antibacterial and antifungal properties of silver nanoparticles, these studies provided important information on the harmful environmental effects of silver nanoparticles on terrestrial plants and aquatic organisms when nanoparticles get into the water. When incubating Daphnia magna (water flea) with different concentrations of nanosilver in pond water, more death of the organism occurred at higher nanosilver concentrations (25 µg/L) and longer nanosilver treatment/exposure times (28 hours). Furthermore, high concentrations of silver nanoparticles (>5,000 µg/L) were more detrimental to Vigna radiata (mung bean) seed germination and seedling formation over a six-day exposure period. The same antibacterial and antifungal properties that make silver nanoparticles useful in consumer products may also cause negative environmental effects if silver nanoparticles enter the water. Therefore, the effects of silver nanoparticles should be carefully weighed against their environmental impact to control hazards associated with widespread use.

Bibliography/Citations:

Bibliography:

  1. Antimicrobial Properties of Nanoparticles. https://nanohub.org/resources/23236/downloads/SilverNanoparticles_Lab_activity.doc
  2. Alexander JW. History of the Medical Use of Silver. Surg Infect. 2009; 10:289-292.
  3. Nanosilver: Naughty or Nice? https://www.sciencenewsforstudents.org/article/nanosilver-naughty-or-nice
  4. https://www.sicencebuddies.org/science-fair-projects/project/can-silver-nanoparticles-neutralize-e-coli-bacteria#procedure
  5. https://www.sicencebuddies.org/science-fair-projects/project/nanosilver-in-consumer-products-affect-pond-life#procedure
  6. Imelda Galvan Marquez et. al. Zinc oxide and silver nanoparticles toxicity in the baker’s yeast, Saccharomyces cerevisiae. PLoS ONE (2018) 13(3).
  7. Matthew Cimitile. Nanoparticles in Sunscreen Damage Microbes. Scientific American, 2009. https://www.scientificamerican.com/article/nanoparticles-in-sunscreen/
  8. Biosafety in Microbiological and Biomedical Laboratories. https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF
  9. Final risk assessment of Escherichia coli k-12 derivatives  https://www.epa.gov/sites/production/files/2015-09/documents/fra004.pdf
  10. Microbiology Techniques and Troubleshooting. https://www.sciencebuddies.org/science-fair-projects/references/microbiology-techniques-troubleshooting
  11. Microorganisms Safety Guide.  https://www.sciencebuddies.org/science-fair-projects/references/microorganisms-safety
  12. https://www.carolina.com/teacher-resrouces/lab-science-classroom-safety-information/10856.com
  13. Care and handling of Daphnia - https://yourtube.com/watch?v=sD9Tg0Qw-Yg; Carolina Biological Supply handling care instructions
  14. Chaloupka et al., Nanosilver as a New Generation of Nanoproduct, Trends in Biotechnology, 2010 Nov;28(11):580-8.
  15. https://www.khanacademy.org/science/high-school-biology/hs-energy-and-transport/hs-osmosis-and-tonicity/v/hypotonic-isotonic-and-hypertonic-solutions-tonicity

Additional Project Information

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Project web pages: -- No webpages provided --
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Research Plan:

MATERIALS:

  • Neutralizing Bacterial Kit, available from Home Science Tools (Item #: SB-BACTUSE), which includes nutrient agar plates, Escherichia coli K-12 culture (LyoQuick freeze dried and ready to reconstitute), 6 mm diameter sterile disks (Item #: LM-STERILE), sterile cotton swabs, nitrile gloves
  • Isopropyl alcohol, 70%
  • Metal forceps
  • Incubator (OppsDecor Egg Incubator, Amazon.com)
  • Candles
  • Baby food jars for silver nanoparticle serial dilutions
  • Distilled water
  • Natural Path Silver Wings Dietary Mineral Supplement, colloidal silver 500 PPM (Amazon.com)
  • Baker’s yeast Saccharomyces cerevisiae (Fleischmann’s Active Dry Yeast Original, ¼ oz packet)
  • Granulated sugar
  • 10X magnifying glass
  • Metric ruler
  • Bleach
  • 10 mL, 25 mL, 100 mL, 500 mL graduated cylinders
  • Petri dishes, polystyrene, 90 X 15 mm, 20 pack (Home Science Tools, Item #: BE-PETRI20)
  • Melt & pour yeast-extract dextrose (YED) media, 350 mL (Carolina Biological Supply Item #: C30910)
  • 2 one-gallon jugs of pond water
  • Daphnia magna cultures (Carolina Biology Supply Company Item # 142331)
  • 1 mL and 3 mL pipets
  • 20 oz clear plastic cups
  • Nature Jim’s Sprouting Mung Bean Seeds (Amazon.com)
  • Consumer products containing silver nanoparticles from Amazon.com: Hylunia Colloidal Silver Mist (10 PPM), Silver Shield Sanitizer Multi-Purpose Hygiene Spray (15-20 PPM), Heritage Store Colloidal Silver Foaming Soap (20 PPM), Silver Biotics Tooth Gel (22 PPM), Curad Germ Shield (55 PPM), Organa Silver Gel (100 PPM), and Silver Lip Balm (2,500 PPM).

PROCEDURES:

I. Evaluation of the Effect of Silver Nanoparticles on the Viability of Escherichia coli using the Kirby-Bauer Antibiotic Testing Method:

  1. Perform a 1:10 serial dilution of the colloidal silver (Natural Path Silver Wings Dietary Mineral Supplement, colloidal silver 500 PPM) in distilled water in clean baby food jars. The concentrations being tested are 500,000 µg/L (undiluted), 50,000 µg/L, 5,000 µg/L, 500 µg/L, 50 µg/L, 5 µg/L and 0 µg/L as negative control. Using a 25 mL graduated cylinder, 22.5 mL of distilled water was poured into jars #2, #3, #4, #5, #6, and #7.  Jar #1 holds the 500,000 µg/L undiluted concentration of colloidal silver. Using a 3 mL pipet, 2.5 mL of the undiluted solution was transferred from jar #1 to jar #2 (50,000 µg/L).  After thorough mixing, 2.5 mL of the solution in jar #2 was transferred to jar #3 (5,000 µg/L) and the process was repeated to achieve the remaining 1:10 serial dilutions for jars #4 (500 µg/L), #5 (50 µg/L), and #6 (5 µg/L).  Jar #7 contained only distilled water.
  2. Sterilize a pair of forceps in 70% isopropyl alcohol and place 6 sterile disks into each baby food jar containing the different concentrations of silver nanoparticles.  The disks were soaked until a later step.
  3. Working in a sterile environment next to lit candles, make bacterial lawns by following the Neutralizing Bacteria Kit manufacturer’s instructions to reconstitute the freeze-dried LyoQuick E. coli bacteria.  Lay out 14 of the nutrient-agar prepared media plates. Place the plates upside down and label the back of each one with the respective concentrations of silver nanoparticles. Dispense 2 drops of the E. coli culture on the center surface of the agar plate and use a sterile cotton swab to spread bacteria around the entire surface of the agar plate. Streak the bacteria by making a vertical line, then spread this left to right and top to bottom, rotate the plate 60 degrees clockwise and repeat the spreading and plate rotation steps 3 more times. Repeat for all 14 plates.
  4. With sterile forceps, evenly place three filter circles from a specific concentration of nanosilver onto the corresponding labeled bacteria plate.  Use 2 bacteria plates for each concentration tested. Tap the sterile circle on the jar’s rim until no drops are coming off the circle (about 6 taps) before placing on the agar plate. Go from the least concentrated to the most concentrated jar to minimize spreading the colloidal silver solutions from a more concentrated jar to a less concentrated one.  It is important not to let the circle move once it is set on the surface.  Using the forceps’ tips, press the surface of the circle once to ensure it is secured onto the agar. 
  5. Secure the lid on each agar plate using a few pieces of clear tape.  Incubate inverted plates in a 37oC incubator with 70% humidity.
  6. Observe each plate at 0 hour, 24 hours, 48 hours, 72 hours and 96 hours. A zone of inhibition around the filter disk suggests that the particular concentration of colloidal silver has antibacterial effects. Use a metric ruler to measure the width (diameter) in millimeters of any zones of inhibition around the filter circles seen on the plates. Place a metric ruler across the zone of inhibition at the widest diameter and measure from one edge of the zone to the other edge.  The filter disk diameter is actually part of the width measurement.  If there is no zone of inhibition at all, report the width number as zero even though the disk itself is around 6 mm in diameter.
  7. Plot the average width measurements as a bar graph to examine which concentrations cause an antibacterial effect. 
  8. Repeat this E. coli experiment using the following consumer products containing silver nanoparticles to assess their antibacterial effects:  Hylunia Colloidal Silver Mist (10 PPM), Silver Shield Sanitizer Multi-Purpose Hygiene Spray (15-20 PPM), Heritage Store Colloidal Silver Foaming Soap (20 PPM), Silver Biotics Tooth Gel (22 PPM), Curad Germ Shield (55 PPM), Organa Silver Gel (100 PPM), and Silver Lip Balm (2,500 PPM). Also include a distilled water negative control. Soak the sterile filter disks in the undiluted product and repeat steps 3 through 7 above to observe any antibacterial effects.
  9. After you finish making observations, sterilize bacteria plates by soaking them in a 10% bleach solution for at least 1-2 hours and discard in the trash. Dilute the colloidal silver solution with tap water until you reach a selected unharmful concentration and pour the dilution into the sink.

Biological Agents and Risk Assessment:  The source of the E. coli K-12 is contained in the Neutralizing Bacteria Kit from Home School Tools.  According to the Public Health Service, “The E. coli K-12 strain is classified as Biosafety Level 1 and is non-pathogenic and as such would not be expected to cause harm to healthy people, animals or to the environment.”  All safety precautions and disposal methods need to be strictly followed.

Safety Precautions: Even though considered safe, E. coli K-12 should be treated at all times as if it were a potential hazard. Experiments should be done with the highest safety precautions and standard sterile technique. Nose and mouth should be kept away from tubes, pipettes, or other tools that come in contact with E. coli cultures and colloidal silver nanoparticles. Gloves should be worn, work surfaces should be disinfected with 70% isopropyl alcohol before and after use, and hands should be washed thoroughly after glove removal. When finished, all materials should be properly disinfected and disposed of safely. Sterilize bacteria plates and pipettes by soaking them in a 10% bleach solution for at least 1-2 hours and discard in the trash. 

II. Evaluation of the Effect of Silver Nanoparticles on the Viability of S. cerevisiae using the Kirby-Bauer Testing Method:

  1. Prior to beginning the experiment, melt yeast-extract dextrose (YED) media (350 mL) in a microwave swirling every 30 seconds to mix.  Working in a sterile environment next to lit candles, pour melted growth media into 90 x 15 mm petri dishes (~25 mL to cover the bottom of the dish). Let petri dishes sit at room temperature for 2 hours to solidify the YED plates.
  2. Perform a 1:10 serial dilution of the colloidal silver (Natural Path Silver Wings Dietary Mineral Supplement, colloidal silver 500 PPM) in distilled water in clean baby food jars. The concentrations being tested are 500,000 µg/L (undiluted), 50,000 µg/L, 5,000 µg/L, 500 µg/L, 50 µg/L, 5 µg/L and 0 µg/L as negative control. Using a 25 mL graduated cylinder, 22.5 mL of distilled water was poured into jars #2, #3, #4, #5, #6, and #7.  Jar #1 holds the 500,000 µg/L undiluted concentration of colloidal silver. Using a 3 mL pipet, 2.5 mL of the undiluted solution was transferred from jar #1 to jar #2 (50,000 µg/L).  After thorough mixing, 2.5 mL of the solution in jar #2 was transferred to jar #3 (5,000 µg/L) and the process was repeated to achieve the remaining 1:10 serial dilutions for jars #4 (500 µg/L), #5 (50 µg/L), and #6 (5 µg/L).  Jar #7 contained only distilled water.
  3. Sterilize a pair of forceps in 70% isopropyl alcohol and place 6 sterile disks into each baby food jar containing the different concentrations of silver nanoparticles.  The disks were soaked until a later step.
  4. Working in a sterile environment next to lit candles, dissolve 1 teaspoon (tsp.) of granulated sugar in ½ cup of warm tap water (43°C to 46°C). When the sugar is fully dissolved, add ½ tsp. of baker’s yeast (Fleischmann’s Active Dry Yeast Original), mix well and let sit for 10 minutes at room temperature. 
  5. Using a sterile 1 mL transfer pipette, transfer 0.1 mL of the yeast extract onto the middle surface of the agar plate and use a sterile cotton swab to spread yeast around the entire surface of the YED agar plate. Streak the yeast by making a vertical line, then spread this left to right and top to bottom, rotate the plate 60 degrees clockwise and repeat the spreading and plate rotation steps 3 more times.  Repeat for all 14 plates.
  6. With sterile forceps, evenly place three filter circles from a specific concentration of nanosilver onto the corresponding labeled yeast plate.  Use 2 yeast plates for each concentration tested. Tap the sterile circle on the jar’s rim until no drops are coming off the circle (about 6 taps) before placing on the YED yeast plate. Go from the least concentrated to the most concentrated jar to minimize spreading the colloidal silver solutions from a more concentrated jar to a less concentrated one.  It is important not to let the circle move once it is set on the surface.  Using the forceps’ tips, press the surface of the circle once to ensure it is secured onto the agar. 
  7. Secure the lid on each agar plate using a few pieces of clear tape. Incubate inverted plates in a 30oC incubator with 70% humidity.
  8. Observe each plate at 0 hour, 24 hours, 48 hours, and 72 hours. A zone of inhibition around the filter disk suggests that the particular concentration of colloidal silver has antifungal effects. Use a metric ruler to measure the width (diameter) in millimeters of any zones of inhibition around the filter circles seen on the plates. Place a metric ruler across the zone of inhibition at the widest diameter and measure from one edge of the zone to the other edge.  The filter disk diameter is actually part of the width measurement.  If there is no zone of inhibition at all, report the width number as zero even though the disk itself is around 6 mm in diameter.
  9. Plot the average width measurements as a bar graph to examine which concentrations cause an antifungal effect. 
  10. Repeat this S. cerevisiae experiment using the following consumer products containing silver nanoparticles to assess their antifungal effects:  Hylunia Colloidal Silver Mist (10 PPM), Silver Shield Sanitizer Multi-Purpose Hygiene Spray (15-20 PPM), Heritage Store Colloidal Silver Foaming Soap (20 PPM), Silver Biotics Tooth Gel (22 PPM), Curad Germ Shield (55 PPM), Organa Silver Gel (100 PPM), and Silver Lip Balm (2,500 PPM). Also include a distilled water negative control. Soak the sterile filter disks in the undiluted product and repeat steps 4 through 10 to observe the antifungal effects.
  11. After you finish making observations, sterilize yeast plates by soaking them in a 10% bleach solution for at least 1-2 hours and discard in the trash. Dilute the colloidal silver solution with tap water until you reach a selected unharmful concentration and pour the dilution into the sink.

Biological Agents and Risk Assessment:  The source of S. cerevisiae (baker’s yeast) is the local grocery store. According to the Public Health Service, “S. cerevisiae is classified as Biosafety Level 1 and is non-pathogenic and as such would not be expected to cause harm to healthy people, animals or to the environment.” All safety precautions and disposal methods need to be strictly followed.

Safety Precautions: Even though considered safe, S. cerevisiae should be treated at all times as if it were a potential hazard. Experiments should be done with the highest safety precautions and standard sterile technique. Nose and mouth should be kept away from tubes, pipettes, or other tools that come in contact with S. cerevisiae cultures and colloidal silver nanoparticles. Gloves should be worn, work surfaces should be disinfected with 70% isopropyl alcohol before and after use, and hands should be washed thoroughly after glove removal. When finished, all materials should be properly disinfected and disposed of safely. Sterilize yeast plates and pipettes by soaking them in a 10% bleach solution for at least 1-2 hours and discard in the trash. 

III. Evaluation of the Effect of Silver Nanoparticles on the Viability of the Aquatic Organism Daphnia magna:

  1. Upon receipt of the Daphnia magna (water flea) culture in the mail from Carolina Biological supply company, immediately unscrew the cap of the container and put it loosely on the top to let oxygen in, which Daphnia need to survive. Let the open jar rest for 24 hours so that the Daphnia can recover from shipping and handling before beginning the experiment. The experiment should be completed within 2 days of receiving the culture.
  2. In clear plastic 20 oz cups, prepare three trials for each concentration of colloidal silver (25 µg/L, 10 µg/L, 5 µg/mL and a negative control 0 µg/L).
  3. Prepare 1,000 µg/L and 500 µg/L nanosilver stock solutions. Using a 1 mL pipet, suck up 1 mL of the Natural Path Silver Wings Dietary Mineral Supplement colloidal silver 500 PPM solution and transfer it into an empty 20 oz plastic cup labeled with 1,000 µg/L.  Measure out 499 mL of pond water with a 500 mL graduated cylinder and add this to the 1 mL nanosilver solution mixing well.  Make a 1:2 dilution of the 1,000 µg/L solution to get a nanosilver concentration of 500 µg/L by adding 250 mL of 1,000 µg/L nanosilver solution to 250 mL pond water and mix well.
  4. Make a 1:100 dilution of the 500 µg/L nanosilver solution to get a concentration of 5 µg/L. Using graduated cylinders, fill 3 cups labeled as 5 µg/L with 495 mL pond water and add 5 mL of the 500 µg/L nanosilver solution to each cup.
  5. Make a 1:100 dilution of the 1,000 µg/L nanosilver solution to get a concentration of 10 µg/L. Fill 3 cups labeled as 10 µg/L with 495 mL pond water and add 5 mL of the 1,000 µg/L nanosilver solution to each cup.
  6. Make a 1:20 dilution of 500 µg/L nanosilver solution to get a concentration of 25 µg/L. Fill 3 cups labeled 25 µg/L with 475 mL pond water and add 25 mL of the 500 µg/L nanosilver solution to each cup.
  7. Add an additional 3 cups with no colloidal silver as a negative control and fill with 500 mL pond water.
  8. Add 10 live Daphnia from the culture to each of the prepared cups using a fresh watering pipet to slowly suck up one Daphnia from the culture. Cut the front end of the pipet at a 45o angle to avoid damaging the live Daphnia during transfer. Transfer as little culture water as possible.
  9. Incubate the Daphnia magna cultures at room temperature during the duration of the experiment.
  10. Wait until 2 hours have passed and then count living and dead daphnia in each cup using a 10X magnifying glass. Live Daphnia will move around the cup and a heartbeat will be detectable.  Dead daphnia do not move, lie on the bottom of the cup or float on the top. Repeat these observations at 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours and 28 hours.
  11. Calculate the average number and the percentage of dead and live daphnia recorded for each nanosilver concentration and the control over exposure time.  Plot a dose-response-curve from the results where the dose (concentration of silver nanoparticles) is depicted on the x-axis and the response (% dead Daphnia) is represented on the y-axis.

Biological Agents and Risk Assessment: The culture of Daphnia magna is isolated from local ponds by Carolina Biological Supply Company.  These organisms fall in the same line as the classification of all microorganisms within the United States of America being governed by the Public Health Service. Carolina Biological Supply Company under these classification regulations classifies this organism as Biosafety Level/Shipping Class I. According to the Public Health Service’s definition for Biosafety Level/Shipping Class I, “this organism is considered to be non-pathogenic and as such would not be expected to cause harm to healthy people, animals or to the environment.” All safety precautions and disposal methods must be strictly followed.

Safety Precautions and Disposal: For maximum safety, maintain proper handling, clean up and disposal of Daphnia magna. Nose and mouth should be kept away from tubes, pipettes or other tools that come in contact with the culture. Gloves must be worn at all times. Add 5 mL of bleach to each cup with collected dead/live Daphnia, mix well and after 20 min pour down the sink. Do not release in the wild. Alternatively, the culture may be used as a food source for fish in a freshwater aquarium. Dilute the nanosilver solution with tap water until you reach an unharmful concentration and pour the dilution into the sink.

IV. Evaluation of the Effect of Silver Nanoparticles on the Germination of the plant Vigna radiata (Mung Bean):

  1. Perform a 1:10 serial dilution of the colloidal silver (Natural Path Silver Wings Dietary Mineral Supplement, colloidal silver 500 PPM) in distilled water in clean baby food jars as described above under bacteria and yeast methods. The concentrations being tested are 500,000 µg/L (undiluted), 50,000 µg/L, 5,000 µg/L, 500 µg/L, 50 µg/L, 5 µg/L and 0 µg/L as negative control.
  2. Add 3 mung bean seeds (Nature Jim’s Sprouting Mung Bean Seeds) to each jar (two jars at each concentration for a total of 6 mung beans per concentration) and observe effects of colloidal silver on seed germination over 6 days.

Safety Precautions and Disposal: Gloves must be worn at all times when handling the silver nanoparticle solutions. Dilute the nanosilver solution with tap water until you reach a selected unharmful concentration and pour the dilution into the sink.  Dispose mung bean seeds and seedlings in the trash.

 

Questions and Answers

1. What was the major objective of your project and what was your plan to achieve it? 

     The major objective of this project was to investigate the antibacterial and antifungal effectiveness of different concentrations of silver nanoparticles using Escherichia coli (a type of bacteria that normally lives in the intestines where it helps the body break down and digest the food we eat) and Saccharomyces cerevisiae (a fungi/yeast used for baking and brewing beer).  Furthermore, this project investigated if consumer products containing silver nanoparticles are also effective antibacterial and antifungal agents. This project also explored what happens if the silver nanoparticles get into the water by investigating how freshwater organisms such as Daphnia magna or water flea and plants such as Vigna radiata (mung bean plant used in Asian cuisine) respond to exposure to different concentrations of nanosilver over time.

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  

Silver has been used throughout history as an antimicrobial agent to fight disease and help the healing process.  There are over 400 consumer products such as sports clothes, socks, bandages, cosmetics, sunscreens, toothpastes, deodorants, sanitizers and food containers that contain tiny silver particles called nanoparticles to help kill bacteria. I wanted to test to see the effectiveness of silver nanoparticles as antibacterial and antifungal agents.  I also wanted to know what happens if silver nanoparticles get into the water? How do freshwater organisms and plants respond to the exposure of different concentrations of nanosilver?  Will the same properties that make silver nanoparticles effective as an antimicrobial agent also be detrimental to ecological health and affect aquatic organisms and terrestrial plants?

       b. Were you trying to solve a problem, answer a question, or test a hypothesis?

My testable questions included: What concentration of silver nanoparticles has a cytotoxic effect on the bacteria Escherichia coli, the fungi Saccharomyces cerevisiae, the aquatic organism Daphnia magna (water flea) and the plant Vigna radiata (mung bean)?  Does silver nanoparticle treatment time have an effect on cytotoxicity?  The hypothesis that I was testing is: If higher concentrations of silver nanoparticles are used to treat E. coli, S. cerevisiae, Daphnia magna, and Vigna radiata, then more cell death will occur because higher concentrations of nanosilver are more cytotoxic. 

2. What were the major tasks you had to perform in order to complete your project?

There were 4 major tasks that I needed to perform to complete the project.  First, I used the Kirby Bauer Method to examine the effect of increasing concentrations of silver nanoparticles on the viability of E. coli and S. cerevisiae. Additionally, I looked at the effect of time and plating density on the viability of these organisms.  Second, I tested the effectiveness of nanosilver-containing consumer products on the viability of E. coli and S. cerevisiae.  Third, I cultured Daphnia magna in pond water containing increasing concentrations of silver nanoparticles over time.  Fourth, I incubated mung been seeds in solutions of increasing concentrations of silver nanoparticles.

3. What is new or novel about your project?

I haven’t seen a science fair project on silver nanoparticles at previous science fairs that I attended and thought that this would be a unique topic to research.  Silver has been used throughout history to fight disease and help the healing process.  I thought that it would be interesting to test nanosilver-containing consumer products for their antimicrobial effectiveness.  When I spoke to several product manufacturers on the phone, I realized that they tested their products for antibacterial properties.  So, then I wondered if these nanoparticles could also do harm to other organisms in the environment if they were exposed by accident. I, therefore, pursued studies of silver nanoparticles and exposure to plants and aquatic organisms.

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before? 

This project was a great learning experience for me.  I have never cultured bacteria or yeast nor have I ever cultured water fleas or worked with mung bean plants.  I learned aseptic techniques and lab methodologies such as the Kirby Bauer Method that will aid me in future projects and lab courses.

       b. Is your project's objective, or the way you implemented it, different from anything you have seen? 

I have never seen a project that compares effectiveness of various consumer products containing silver nanoparticles on yeast and bacteria cell killing.

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?  I searched through science fair topics, looked on the internet and contacted vendors of products that contain silver nanoparticles.

4. What was the most challenging part of completing your project?

The most challenging part of this project for me was the culture of the E. coli, S. cerevisiae and Daphnia magna. I previously had no experience with aseptic technique or handling these organisms.  I had to learn and study the safe handling of these organisms.

      a. What problems did you encounter, and how did you overcome them?

The biggest challenge was lyophilizing the E. coli bacteria that came with the Neutralizing Bacteria kit.  I applied too much pressure on the ampule causing the bacteria dispenser to break.  As a result, I did not have enough bacteria to test all the conditions.  Fortunately, the vendor replaced the agar plates and E. coli and I had enough materials to complete 6 trials.  Another problem that I encountered was the culture of the Daphnia magna (water fleas).  The vendor noted that culture should be used within two days of receiving the culture.  I indeed observed that the untreated culture started to die naturally after 32 hours so I had to stop the experiment.

      b. What did you learn from overcoming these problems?

I learned the tricks to lyophilizing and efficiently plating bacteria in a sterile environment.

5. If you were going to do this project again, are there any things you would you do differently the next time?

I would have liked to have had access to a higher magnification microscope so that I could observe the antimicrobial effects of silver nanoparticles at earlier timepoints (< 24 hours).  It would also be great to observe the Daphnia magna under higher magnification. I could then plot the rate of the heartbeat after exposure to different concentrations of nanosilver.  I would also like to see if I could prolong the culture for the Daphnia magna so that I could do longer treatments times.  I would like to go higher in the nanosilver concentrations for the Daphnia magna treatment.

6. Did working on this project give you any ideas for other projects? 

Yes, by working on this project, I had the following ideas for future studies:

  • This project can be expanded to include a larger variety of bacteria and yeast strains and a broader representation of terrestrial plants and aquatic plants (i.e., duck weed or Elodea, etc.) and animals (i.e., snails or fish, etc.).
  • It would also be interesting to study how different sizes, shapes, surface areas, and coatings of the silver nanoparticles affect their cytotoxic properties.
  • Colloidal silver can be isolated from consumer products known to have silver nanoparticles in them to test for its cytotoxicity. Furthermore, since consumer projects may have multiple components that could cause antibacterial or antifungal effects, one could ask vendors to send a sample of the individual product components to test which individual component causes the most cytotoxic effect.
  • One can also inquire if different life stages of Daphnia or other aquatic organisms (baby, juvenile or adult) react the same way to nanosilver.
  • It would also be very interesting to test the effects of silver nanoparticles on human-derived cell lines in culture.

7. How did COVID-19 affect the completion of your project?

COVID-19 did not have any serious effects on the completion of my project.  I only experienced shipping delays on materials needed to conduct the experiments.