Related Links

• Science Fair Project Guide

---

• Microorganisms Safety Guide
• Projects Involving Potentially Hazardous Biological Agents

Project Summary

Difficulty	7
Time required	Long (a couple of weeks)
Prerequisites	None
Material Availability	This science fair project requires access to some laboratory equipment, such as a 37°C incubator, a Bunsen burner, as well as some specialty reagents, which can be ordered online. It also requires several pieces of jewelry, including at least one 14-karat gold piece. See the Materials and Equipment list for more details.
Cost	Average ($50 - $100)
Safety	This science fair project involves the use of the bacterium E. coli. Standard microbiology and bacterial safety guidelines should be followed. See the Microorganisms Safety Guide for more details. Projects using bacteria may require pre-approval from your science fair's Scientific Review Committee. To maintain a sterile environment, you will be working near a lighted Bunsen burner. Keep all flammables, including hair and clothing, away from the flame.

Share this Project Idea! |

Donate to Science Buddies

Sponsor

Sponsored by a generous grant from The Abbott Fund

Abbott Fund Promotes Science

Science Literacy Today Endless Opportunities Tomorrow

www.abbottfund.org

Abstract

Objective

Determine if the antimicrobial properties of metals are a good test for whether or not a piece of jewelry is made out of real gold.

Introduction

Have you ever looked at a piece of gold jewelry and wondered "Is it real?" To answer that question, you might first have to decide on the definition of "real." Gold jewelry is usually described in karats (kt). 24-kt gold is pure gold. While theoretically it is 100% gold, most countries allow a 1% margin for impurities. Pure gold is relatively soft and easily dented, which means that jewelry made from pure gold is easier to accidentally damage. So gold is often mixed with a variety of metals, including copper, zinc, and silver, to make jewelry of different physical strengths, karats, colors, and monetary values. There are also cultural influences over what people think of as "real" gold jewelry. In most Asian countries, "real" gold jewelry is usually 21-24 kt. In the United States, "real" gold jewelry is normally 10 kt, 14 kt, or 18 kt. However, looks are rarely a good way to judge if a piece of jewelry contains a significant proportion of gold. Pieces that are less than 9 kt (the international cutoff for "real" gold jewelry), or that are gold-plated (meaning they have a thin layer of gold put over the surface of a less-expensive metal), or that are even made of brass, can still have the shiny appearance of yellow-gold.

So, how can we differentiate between real gold jewelry and all the others? Because gold is a natural element (number 79, atomic symbol Au on the periodic table of elements), it has specific chemical and physical properties. Jewelers and laboratories that specialize in testing metal use these properties to decide whether a sample is made of gold, and what the level of purity, or karatage, of the gold is. These tests include properties like how the sample scatters light from X-rays, how heavy the ions released from the sample are, and the exact weight of the sample once it is purified.

Figure 1. Looks alone aren't enough to tell which of these pieces of jewelry are gold and which are merely gold-plated, or other metals altogether. From left to right, these pieces are made up of other metal alloys, gold plating, and 24-kt gold.

It turns out that in addition to these physical and chemical properties, gold has a biochemical property: it is toxic to some life forms. Although it is completely harmless to humans and other mammals, gold can be harmful to bacteria, algae, molds, and fungi. Some other metals, including nickel, copper, cadmium, and mercury, also have this property. Collectively, metals capable of causing harm to organisms are often referred to as heavy metals or toxic metals and many of them, like mercury and cadmium, are harmful to humans, too. Usually, it is not the metal that is toxic, but the ions it forms when in contact with water. An ion is an atom or molecule in which the total number of electrons does not equal the number of protons, which gives it a net negative or positive charge. These ions are easily transported into tissues and cells where they interfere with normal functions. The exact mechanism of their interference depends on the type of metal ion, and on the organism under attack. This toxic effect of metal ions, even in very low concentrations, on bacteria is called the oligodynamic effect. The word oligodynamic comes from the Greek words oligos, meaning "few," and dyanmis, meaning "force."

In this science fair project, you will investigate whether the oligodynamic effect of gold on bacteria can be used to determine whether a piece of jewelry is composed of gold, is just gold-plated, or is not gold at all. To do this, you will use a harmless strain of the bacterium Escherichia coli (E. coli). You'll spread the bacteria uniformly across four agar plates: one with no jewelry, to serve as a control, one with gold jewelry, one with gold-plated jewelry, and the last with non-gold jewelry. In the plates containing jewelry, the water in the agar will combine with the metal to form metal ions. Don't worry, it won't harm your jewelry! The amount of metal that is converted into metal ions is so small you wouldn't even be able to measure the loss using the most sensitive of scales. These metal ions will diffuse out from the jewelry into the agar where they can be absorbed by the bacteria. The concentration of metal ions will be highest right next to the jewelry and gradually reduce as you move out in concentric circles from the jewelry. If the metal ions produced have an oligodynamic effect, meaning if they're toxic to the E. coli, the bacteria will not grow where the ions are present. The areas without bacterial growth are referred to as zones of inhibition and are characterized by a clear circle around the jewelry in an otherwise contiguous lawn of bacteria. The larger the zone of inhibition, as measured by the diameter of the clear circle around the jewelry, the greater the oligodynamic effect. Will the different types of jewelry (gold, gold-plated, and non-gold) exhibit different degrees of oligodynamic effect? Can zones of inhibition be used to determine whether a piece of jewelry is "real" gold? Try it and find out for yourself!

Figure 2. This photo shows zones of inhibition, the clear circles surrounding the white disks in what is otherwise a lawn of bacteria, for a Kirby-Bauer disk-diffusion assay. The procedure for this science fair project is an adapted version of that assay. In both cases, the size of each zone of inhibition is measured with a ruler, and the data is recorded in a lab notebook. (D.M. Rollins and S.W. Joseph, 2000.)

Terms, Concepts and Questions to Start Background Research

• Gold
• Karat (kt)
• Gold-plated
• Brass
• Periodic table of elements
• Biochemical
• Heavy metals
• Toxic metals
• Ion
• Oligodynamic effect
• Escherichia coli (E. coli)
• Positive control
• Zone of inhibition
• Kirby-Bauer disk-diffusion method

Questions

• If a necklace is described as being 14-kt gold, what does this mean?
• What metals is gold commonly mixed with? What effects do these various metals have on the gold?
• What methods are used for telling if an object is made of gold or not?
• Which metals are considered to be toxic to humans?
• How do metal ions form?
• The procedure in this science fair project is an adaptation of the Kirby-Bauer disk-diffusion method. What is this method and for what is it typically used? How is this project's procedure similar, and how does it differ?

Bibliography

Information about gold and the karatage system is available at this website:

• World Gold Council. (2009). The Caratage (Karatage) System for Gold Jewellery. Retrieved November 9, 2009, from http://www.utilisegold.com/jewellery_technology/caratage/

This resource discusses toxic metals:

• Occupational Safety & Health Administration. (2009, June 2). Toxic Metals. Retrieved November 9, 2009, from http://www.osha.gov/SLTC/metalsheavy/index.html

An overview of the oligodynamic effect is given here:

• Wikipedia Contributors. (2009, September 15). Oligodynamic Effect. Wikipedia: The Free Encyclopedia. Retrieved November 9, 2009, from http://en.wikipedia.org/w/index.php?title=Oligodynamic_effect&oldid=314201397

This animation shows the setup and use of the Kirby-Bauer disk-diffusion method in a clinical setting. The bacterial growth conditions are different in the science fair project below, but the basic concept is the same as in this animation. The last animation section covers how to interpret the results of the test, which will be particularly useful for your science fair project.

• Shoeb, H. (2008, January 30). Antimicrobial Susceptibility Testing (Kirby-Bauer) Animation. Retrieved November 9, 2009, from the American Society for Microbiology MicrobeLibrary website: http://www.microbelibrary.org/asmonly/details.asp?id=2754&Lang

This website explains the Kirby-Bauer Disk-Diffusion method:

• Rollins, D.M. and Joseph, S.W. (2000, August). Antibiotic Disk Susceptibilities: Kirby-Bauer Disk-Diffusion Method. Retrieved November 9, 2009, from http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm

Materials and Equipment

Note: If you are carrying out this experiment in a school laboratory, which is recommended, some of the materials and equipment listed below may be more readily accessible.

• E. coli, available from online suppliers, such as:
• Sargent-Welch at sargentwelch.com, catalog #WL23601. Note: Sargent-Welch does not sell bacterial cultures to the general public. You will need your school teacher, or a sponsor from a research laboratory or biotech company, to order from this company.
• Carolina Biological at www.carolina.com, catalog #124500. Note: Carolina Biological will sell this bacteria directly to the general public.
• Nutrient broth, available from online suppliers such as Sargent-Welch, catalog # WL54480-21P or Carolina Biological, catalog # 826150
• Nutrient agar plates (12); available from online suppliers, such as Sargent-Welch, catalog # WL54480-20P or Carolina Biological, catalog # 821862
• Permanent marker
• Bunsen burner
• Micropipette (P1000) and tips, or sterile transfer pipette calibrated to 1 mL, available online from Carolina Biological, catalog #214551
• Access to a refrigerator
• Bacterial spreader, or sterile cotton swabs (from a previously unopened package counts as sterile)
• Beaker
• 70% ethanol
• Latex gloves (6 pairs). Can be purchased at a drug store or pharmacy. If you are allergic to latex, use vinyl gloves.
• Mild dish detergent
• Clean dish cloth
• Jewelry (3 pieces). Should all be similar sizes and shapes—the larger the pieces, the easier the results will be to interpret. Flexible link bracelets, necklaces, or anklets work best. The three jewelry types you need are as follows:
• 14-kt gold or higher (1). Gold jewelry sold in the United States is stamped with a karat mark which indicates the karatage; a 14-kt necklace would have a "14K" stamped somewhere on the surface of the necklace.
• Gold-plated (1), sometimes referred to in commercial jewelry descriptions as "gold over"
• Not gold, but some other "gold-colored" metal alloy, like brass (1), sometimes referred to as "gold toned" in commercial jewelry descriptions
• 37°C incubator.
• Note: If you have access to one, it is recommended that you use a 37°C incubator to grow your plates in, but the project can be done without one. See the Experimental Procedure for more details.
• Ruler with millimeter (mm) markings
• Lab notebook

Disclaimer: Science Buddies occasionally provides information (such as part numbers, supplier names, and supplier weblinks) to assist our users in locating specialty items for individual projects. The information is provided solely as a convenience to our users. We do our best to make sure that part numbers and descriptions are accurate when first listed. However, since part numbers do change as items are obsoleted or improved, please send us an email if you run across any parts that are no longer available. We also do our best to make sure that any listed supplier provides prompt, courteous service. Science Buddies receives no consideration, financial or otherwise, from suppliers for these listings. (The sole exception is any Amazon.com or Barnes&Noble.com link.) If you have any comments (positive or negative) related to purchases you've made for science fair projects from recommendations on our site, please let us know. Write to us at scibuddy@sciencebuddies.org.

Experimental Procedure

• The bacteria used in this science fair project, E. coli, is a biosafety level 1 bacteria and thus considered to be very safe for healthy humans to handle. However, many science fairs require students who use bacteria in their projects to fill out special risk-assessment forms and to conduct their experiments in the confines of a school laboratory. Consult with your teacher, or an official from your local science fair before you begin. You might also find our information for Projects Involving Potentially Hazardous Biological Agents useful in determining what paperwork you might need to fill out.
• While E. coli is not considered a biohazardous or dangerous bacterium, it is important to always properly clean and dispose of bacteria and supplies that come in contact with it. See the Bacterial Safety guidelines below for more details about how to handle bacterial cleanup and waste.

Getting the Jewelery Ready

You'll need to clean the jewelry before starting this science fair project. This will ensure that the surface of the jewelry is free from contaminating bacteria, and that the metal, gold, or otherwise, is in direct contact with the agar plates, rather than shielded by dust or oil.

1. While wearing latex gloves, wash the jewelry in mild dish detergent with warm water. Rinse it well.
2. Gently dry the jewelry with a clean dish cloth and set it aside.

Preparing the Test Plates

1. If you ordered your E. coli stock from Carolina Biological, it will arrive as a culture growing on a slab of agar in a tube. Media (nutrient broth) needs to be added so that you have a liquid culture to use. If you ordered your E. coli stock from Sargent-Welch, it came as a liquid culture, so skip to step 2 in this section.
1. Using a sterile pipette, add 3 mL of nutrient broth to the agar slab in the tube.
2. Re-cap the tube and shake it gently. Allow it to sit for 30 minutes and proceed to step 2 while you wait.
2. Label four nutrient agar plates with the permanent marker.
1. Petri dishes should always be labeled in permanent marker and on the bottom of the plate. Labeling the lid is not sufficient, as the lid is removable and might accidentally get swapped with another plate.
2. One of the plates will be a control to make sure your bacterial lawns grow well and evenly. Label this plate: Control.
3. The other three plates will be your actual gold jewelry experiment plates—one for each piece of jewelry. Label them:
• Non-gold
• Gold plated
• Gold, and the proper karatage. For example: 14-kt Gold
3. To make your bacterial lawns, you will need to work in a sterile environment. Work next to a lighted Bunsen burner, but be careful around the flame. If you have long hair, tie it back. Do not wear flowing sleeves, and avoid reaching across the flame.
1. Take your E. coli culture from step 1 and gently shake it to evenly distribute the bacteria. Using a micropipette or sterile transfer pipette, add 500 µl of the E. coli culture to the surface of an agar plate. Try to put the culture in the center of the plate. Refrigerate the remainder of the culture for repeats.
2. Sterilize the bacterial spreader by dipping it in a beaker containing approximately 30 mL of 70 percent ethanol, and then holding the spreader in the flame of the Bunsen burner for 10 seconds. Caution: You do not want the beaker of ethanol to catch on fire; ethanol is highly flammable, which is why it is used here in combination with the flame to sterilize the spreader. To avoid potential accidents, keep the beaker of ethanol on the opposite side of your workspace from the Bunsen burner.
• Note: If you do not have a bacterial spreader, use sterile cotton swabs to spread the bacteria across the surface of the agar plates. You may need to add more of the E. coli culture if the swabs are soaking up too much of it.
3. Hold the spreader away from the flame for an additional 30 seconds to let it cool. If the metal is too hot when it touches the bacterial cells, it will kill them.
4. Using the spreader, spread the drops of E. coli culture uniformly around the agar plate with E. coli on it.
• Start by gently touching the spreader to an area of the plate far away from the drops of bacteria. This way, if the spreader is too hot, you won't kill the bacteria.
• Move the spreader to the center of the plate where the bacteria are. Spread the bacteria across the whole surface of the plate using up-and-down motions.
• Turn the plate 90 degrees and repeat the spreading motion.
• Keep turning the plate by 90 degrees and spreading until you've been around the whole plate (a total of four 90-degree turns).
5. Remember to put the lid on the agar plate as soon as you are done to prevent other bacteria and contaminants from floating in.
6. Repeat the pipetting, sterilization, and bacterial spreading steps for all four of your agar plates.
4. After you've spread all your agar plates, wait 5 minutes for the surface of the plates to dry. Remember, keep the plates covered.
5. Now you're ready to place the jewelry on the plates.
1. Do not add anything to the Control plate.
2. Put on a fresh pair of latex glove. Add the corresponding piece of jewelry to each of the plates labeled: Gold, and Non-gold.
• Each piece of jewelry should be placed in the middle of the corresponding plate.
• Arrange each piece of jewelry in a manner so that the maximum amount of jewelry surface area is in contact with the agar plate. If the jewelry is made of flexible links, coil it so that it makes a continuous circle on the plate. See Figure 3, below, for a visual example.
• Try to make each piece of jewelry the same size and shape, preferably a circle, on the plate.

Figure 3. Each piece of jewelry should be arranged on its own plate. If possible, coil the jewelry to form a circle with the maximum amount of surface area in contact with the agar plate.

6. Re-cover and incubate the plates at 37°C for 48 hours. Make sure to invert the plates (lid-side down, agar-side up) so that any water condensation does not fall onto your bacterial lawn.
1. Note: If you do not have access to a 37°C incubator, you can grow the bacteria at room temperature. Keep the plates away from direct sunlight, but in a warm part of the house. For example, you may want to keep them in a plastic bag (to protect them from dust) next to a heating vent or the clothes dryer. The incubation time will be longer than in an incubator. Start checking your control plates after 72–96 hours of growing time.

Measuring Zones of Inhibition

1. After 48 hours of incubation (72–96 hours if you are not using a 37°C incubator), examine your plates (keep the lids on while you do this).
1. Do you see a lawn of bacteria on the Control plate? If not, incubate the plates for an additional 24–48 hours until there is a decent bacterial lawn.
.
2. The Control plate should show relatively uniform lawns. If you see dense bacterial growth in some areas and swatches of light or no bacterial growth in other areas, then your bacteria-spreading technique needs improvement. You'll need to repeat the experiment again, paying special attention to spreading the E. coli culture across the plates to get reliable data.
3. If the metal in the jewelry has an oligodynamic effect, you should see zones of inhibition around the jewelry. The edges of the clear zone should be a relatively uniform distance away from the edges of the metal.
2. Using a ruler, measure, in millimeters (mm), the zones of inhibition.
1. If the three pieces of jewelry are all arranged in circles of similar size, measure the diameter of the clear zone.
2. If the jewelry is not circular, measure the distance from the edge of the clear zone to the first edge of the jewelry. Make several measurements around the piece of jewelry and average the measurements to get one representative data point.
3. Record all your data in a data table in your lab notebook.

Repeating the Experiment

1. To ensure that your observations are accurate and repeatable, carry out the experiment two more times.
1. If possible, use a new culture each time.
2. If a new culture is not available, remove the old E. coli culture from the refrigerator, add 3 mL of nutrient broth. Re-cap and gently shake tube. Leave tube at 37°C for one hour. Use this culture as your starting point for creating the bacterial lawn.

Analyzing the Data

1. Are the sizes of the zones of inhibition consistent across your replicates? Calculate the average and standard deviation for each piece of jewelry you tested. For more information about calculating and interpreting standard deviations consult the Science Buddies guide to Variance and Standard Deviation.
2. How do the average zones of inhibition compare for each type of jewelry? Did one type have a larger zone of inhibition than another? Do any of them show an oligodynamic effect?

Bacterial Safety

Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders. You can also see the Microorganisms Safety Guide for more details.

• Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
• Make sure to wash your hands thoroughly after handling bacteria.
• Proper Disposal of Bacterial Cultures
• Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1-2 hours.
• Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
• After bleach treatment is completed, these items can be placed in your normal household garbage.
• Cleaning Your Work Area
• At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
• Be aware of the possible hazards of disinfectants and use them carefully.

Variations

• Do other metals have an oligodynamic effect? Are they greater or smaller than gold's? Devise an experiment to find out.
• If you see a difference between the gold, gold-plated, and non-gold jewelry pieces in the experiment above, try using your observations as a way to determine whether other pieces of jewelry are real gold or not. Hint: consider using a "blind" test method.
• The science fair project above does not distinguish between the metals being bacteriocidal or bacteriostatic. Things that are bacteriocidal kill the bacteria, whereas those that are bacteriostatic temporarily inhibit bacterial growth. If you observe an oligodynamic effect, devise a test to determine if the metal ions are bacteriocidal or bacteriostatic.

• For more science project ideas in this area of science, see Microbiology Project Ideas.

Credits

Veselin Dobrev, OSI Pharmaceuticals, Inc.

Edited by Sandra Slutz, PhD, Science Buddies

Last edit date: 2009-11-12 12:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Microbiology.

	Biological Technician What do the sequencing of the human genome, the annual production of millions of units of life-saving vaccines, and the creation of new drought-tolerant rice varieties have in common? They were all accomplished through the hard work of biological technicians. Scientists may come up with the overarching plans, but the day-to-day labor behind biotech advances is often the work of skilled biological technicians.			Materials Scientist and Engineer What makes it possible to create high-technology objects like computers and sports gear? It's the materials inside those products. Materials scientists and engineers develop materials, like metals, ceramics, polymers, and composites, that other engineers need for their designs. Materials scientists and engineers think atomically (meaning they understand things at the nanoscale level), but they design microscopically (at the level of a microscope), and their materials are used macroscopically (at the level the eye can see). From heat shields in space, prosthetic limbs, semiconductors, and sunscreens to snowboards, race cars, hard drives, and baking dishes, materials scientists and engineers make the materials that make life better.
	Microbiologist Microorganisms (bacteria, viruses, algae, and fungi) are the most common life-forms on Earth. They help us digest nutrients; make foods like yogurt, bread, and olives; and create antibiotics. Some microbes also cause diseases. Microbiologists study the growth, structure, development, and general characteristics of microorganisms to promote health, industry, and a basic understanding of cellular functions.

Join Science Buddies Become a Science Buddies member! It's free! As a member you will be the first to receive our new and innovative project ideas, news about upcoming science competitions, science fair tips, and information on other science related initiatives. Support Science Buddies If this website has helped you, won't you consider a small gift so we may continue developing resources to help teachers and students?