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• Projects Involving Potentially Hazardous Biological Agents

Project Summary

Difficulty	7  –  8
Time required	Long (a couple of weeks)
Prerequisites	None
Material Availability	This science project requires access to some laboratory equipment, such as a 37°C incubator, as well as some specialty reagents, which can be ordered online. See the Materials and Equipment list for more details.
Cost	High ($100 - $150)
Safety	This science 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. 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.

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Abstract

Objective

In this science fair project you will determine which over-the-counter acne medications are the most effective at stopping the proliferation of E. coli.

Introduction

Have you ever had acne? Almost everyone has! Clinicians estimate that nearly 100% of the population will have acne at some time during their lives, usually as adolescents. Acne forms in pilosebaceous units, which are compromised of a hair; a follicle, or shaft in the skin through which the hair grows; and a sebaceous gland. The sebaceous gland produces an oily substance, sebum, which protects the skin and hair, making them waterproof and preventing them from drying out. Dead keratinocytes, or skin cells, and excess sebum can clog the pilosebaceous unit. The clogged follicles are ideal environments for the bacteria that normally live benignly on the skin's surface, to proliferate. This proliferation causes local inflammation and the visual signs are referred to as acne.

Figure 1. This diagram shows the components of a normal pilosebaceous unit, including the follicle and sebaceous gland. (NIAMS, 2006.)

One way to treat acne is to stop the proliferation of the bacteria. In this science fair project, you will test a variety of over-the-counter acne medications to determine which is the most effective at preventing bacterial proliferation. Although Propionibacterium acnes (P. acnes ) is the bacteria which most frequently contributes to acne, in this science fair project you’ll be using the bacterium Escherichia coli (E. coli). E. coli is safer and easier to work with. You'll use the Kirby-Bauer disk-diffusion method to measure the effectiveness of each acne medication. With this technique, bacteria is uniformly spread across an agar plate, then sterile disks, each coated with a compound (in this case, medication) to be tested, are placed on the surface of the agar. The compounds diffuse out from the disk into the agar. The concentration of the compound is highest right next to the disk and gradually reduces as you move out in concentric circles from the disk. If a compound is effective against the bacteria at a certain concentration, no colonies will grow in the area of the plate where that compound is equal to or higher than the effective concentration. The areas without bacterial growth are called zones of inhibition and are characterized by a clear circle around the disk in an otherwise contiguous lawn of bacteria. The larger the zone of inhibition is, as measured by the diameter of the clear circle, the more effective the compound.

Figure 2. The zones of inhibition are the clear circles surrounding the disks in the bacterial lawn. The size of each zone 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

• Acne
• Pilosebaceous unit
• Follicle
• Sebaceous gland
• Sebum
• Keratinocyte
• Propionibacterium acnes (P. acnes)
• Escherichia coli (E. coli)
• Kirby-Bauer disk-diffusion method
• Zone of inhibition
• Anaerobe

Questions

• How does acne develop?
• What are the different types of lesions that make up acne?
• What are the causes of acne?
• Can you name a few types of treatments and medications are available to treat acne?
• What type of bacteria is E. coli? What kind of growth conditions does it require? How about P. acnes?
• What are the real-world applications of the Kirby-Bauer disk-diffusion method?

Bibliography

Here are some resources about acne to get your background research started.

• American Academy of Dermatology. (2008). AcneNet: A comprehensive online acne information resource. Retrieved April 9, 2008 from http://www.skincarephysicians.com/acnenet/index.html
• National Institute of Arthritis and Musculoskeletal and Skin Diseases. (2006, January). Questions and Answers About Acne. Retrieved April 9, 2008 from http://www.niams.nih.gov/Health_Info/Acne/default.asp

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

• Rollins, D.M. and Joseph, S.W. (2000, August). Antibiotic Disk Susceptibilities: Kirby-Bauer Disk-Diffusion Method. Retrieved April 17, 2008 from http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm

Materials and Equipment

Note: 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. Consult with your teacher, or an official from your local science fair. You may also find our information for Projects Involving Potentially Hazardous Biological Agents useful in determining what paperwork you might need to fill out.

• E. coli, available from online suppliers such as:
  • Sargent-Welch, catalog #WL23601. Note: Sargent-Welsh 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, catalog #124500. Note: Carolina Biological will sell this bacteria directly to the general public, but since the bacteria does not come as a liquid culture you will also need to order Nutrient Broth, catalog # 826150.
• Micropipette (P1000) and tips, or sterile transfer pipette calibrated to 1 mL, available online from Carolina Biological, catalog #214551.
• Bacterial spreader, or sterile cotton swabs (from a previously unopened package counts as sterile)
• Nutrient agar plates, available from online suppliers such as Sargent-Welch, catalog # WL54480-20P or Carolina Biological, catalog # 821862. The number of plates you need depends on how many medications you choose to test. See the table below for details.

  # of Medications to be Tested	# of Plates Needed
  3	6
  4–6	9
  7–9	12

• Permanent marker
• Bunsen burner
• Beaker
• 70% ethanol
• Bacterial spreader
• Sterile disks, available from online suppliers such as Sargent-Welch , catalog # WL55135-A, or Carolina Biological, catalog # 805091
• Sterile forceps or tweezers
  • To ensure that they are sterile, autoclave them. If autoclaving is not an option, place them in boiling water for 15 minutes.
• Small sterile dishes (one for each medication being tested)
• Sterile water
  • Water should be autoclaved to sterilize it. If autoclaving is not an option, boil the water for 15 minutes.
• Acne medications (minimum of 3 different medications)
• Sterile cotton swabs (from a previously unopened package counts as sterile)
• Sterile cotton balls (from a previously unopened package counts as sterile)
• 37°C incubator. Note: If you have access to one, it is recommended that you use a 37C incubator to grow your plates in – but the project can be done without one, see Experimental Procedure for more details.
• Lab notebook
• Ruler with millimeter (mm) markings

Experimental Procedure

This science fair project involves the use of the bacteria E. coli. While E. coli is not considered a biohazardous or dangerous bacteria, 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 on how to handle bacterial cleanup and waste.

Preparing Plates for Acne Medication Effectiveness Test

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 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, skip to step 2 in this section.
   1. Using a sterile pipette, add 7 mL of nutrient broth to the agar slab in the tube.
   2. Re-cap the tube and shake gently, allow to sit for 30 minutes, proceed to step 2 while you wait.
2. Label your nutrient agar plates.
   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. Three of the plates are controls to make sure your bacterial lawns grow well and evenly. Label these plates: Control #1, Control #2, and Control #3.
   3. The other plates are your actual acne medication trial plates. Divide each plate into four equally sized quadrants with the permanent marker. See Figure 3 below for an example.
      • Label one quadrant on each plate Water. This will be your negative control.
      • The other three quadrants should be labeled with the names of three different acne medications you are testing.
      • Each acne medication will be tested three times, but it should be tested on three different plates so that the maximum amount of experimental error is accounted for in your observations. Do not put all three replicates of an acne medication on the same plate.

        So, for every three acne medications you will label three plates where the quadrants read:

        1. Water, Acne medication 1A, Acne medication 2A, Acne medication 3A
        2. Water, Acne medication 1B, Acne medication 2B, Acne medication 3B
        3. Water, Acne medication 1C, Acne medication 2C, Acne medication 3C

   Figure 3. This photo shows how your agar plates should be divided into quadrants and labeled.

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.
   2. Sterilize your bacterial spreader by dipping it in a beaker containing approximately 30 mL of 70% 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.
   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.
      • 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 of your agar plates.
   7. 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.
4. After you've spread all your agar plates, wait 5 minutes for the surface of the plates to dry. Remember, keep those plates covered!
5. Now you're ready to apply the negative control (sterile water) and medications to the agar plates.
   1. Do not add any substances to the three control plates.
   2. The remaining plates will each get a negative control—a disk dipped in sterile water—and up to three disks dipped in different acne medications.
      • Put each of your acne medications into its own small sterile dish.
      • Using sterile forceps, dip a sterile disk into the first acne medication you want to test. If the medication is a liquid, gently shake the disk to remove excess liquid. If the medication is a cream or gel, use a sterile cotton swab to remove excess medication from the disk.
      • Make sure that the label on the underside of the plate matches the medication you are testing. Place the disk in the middle of the appropriate quadrant on one of the agar plates. See Figure 4 for an example. Using the forceps, press down gently to ensure good contact between the agar plate and the disk.
      • Clean and re-sterilize the forceps between each use (i.e. after you have placed one disk, but before you place the next disk) by wiping the forceps with a sterile cotton ball that has been wet with 70% ethanol.
      • Repeat Step 4b so that you have three plates for each acne medication.
      • Using the same technique, dip a disk in the sterile water. Apply one disk of sterile water to each agar plate. This will serve as a negative control. Sterile water should not create zones of inhibition; if zones do appear, you have a cross-contamination problem.

   Figure 4. The experimental plates should look similar to this once you have applied the medicated disks to the agar.

6. 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 three control plates you made? If not, incubate the plates for an additional 24-48 hours until there is a decent bacterial lawn.
   2. The three control plates 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 medications you chose are effective at preventing E. coli growth, you should see zones of inhibition around the medication disks. The clear zones around each disk should be circular with a relatively uniform diameter since diffusion of the substances through the agar is the same in every direction. If the zones of inhibition do not have uniform diameters, there are two possible problems:
      • The medication was not distributed evenly across the disk.
      • There was poor contact between the disk and the agar.
      • If the variability is particularly high, you might want to repeat the experiment, paying careful attention to how you apply the acne medications.
   4. There should be no zone of inhibition around your sterile water controls.
2. Using a ruler, measure, in millimeters (mm), the diameter of the zone of inhibition around each medication disk. Do not take the lids off to do this. Instead, invert the plates and measure with the agar side closest to you. Record all your data in a data table in your lab notebook. You will have three measurements for each medication, each from a different plate.
3. Analyze your data. Are the sizes of the zones of inhibition consistent across your replicates? Calculate the average and standard deviation for the medications you tested. Which acne medication was most effective; which was least effective?
4. If you cross-compare the active ingredients in each medication with the results of your experiment, do you see any trends? Are there certain ingredients that seem to contribute significantly to preventing E. coli growth? If so, consider designing an additional experiment to test your new hypothesis.

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

• The science fair project above does not distinguish between bacteriocidal and bacteriostatic compounds. Those that are bacteriocidal kill the bacteria, whereas those that are bacteriostatic temporarily inhibit bacterial growth. Devise an experiment to test whether the acne medications are bacteriocidal or bacteriostatic. You should only test the medications which proved effective against E. coli (i.e. those with the largest zones of inhibition).
• Do any of the zones of inhibition have one or a few colonies within an otherwise bacterial-growth-free area? If so, think about what could be different about these colonies. Design an experiment to test your hypothesis.
• Which antibiotics is E. coli susceptible to? Design an experiment to find out. Note: Antibiotic sensitivity disks are available from online suppliers such as Sargent-Welch and Carolina Biological.

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

Credits

Sandra Slutz, PhD, Science Buddies

Last edit date: 2008-04-23 22:00:00

Career Focus

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

	Epidemiologist Do you like a good mystery? Well, an epidemiologist’s job is all about solving mysteries—medical mysteries—but instead of figuring out “who done it” like a police detective would, they figure out “what caused it.” They find relationships between a medical condition and things like human behavior, environmental toxins, genes, medical treatments, other diseases, and geographical location. For example, they ask questions like what causes multiple sclerosis? How can we prevent brain cancer? What is the “vector” or animal that is transmitting the hantavirus? Which populations are most at risk from a new flu virus? Epidemiologists work to answer these and thousands of other questions in an effort to reduce public health risks. Their work has the potential to save millions of lives.			Agricultural Inspector Who works to protect the public health from food-borne illnesses? Agricultural inspectors. Everyone needs to eat, and agricultural inspectors work to ensure the quality and safety of the food supply to determine if they are in compliance. They also inspect farms, businesses, and food-processing plants to determine if they are in compliance with government food regulations and laws.
	Water & Liquid Waste Treatment Plant & System Operator Have you ever wondered what happens to that soapy water from your kitchen sink or laundry room washer, or the waste water from your bathroom? What about the water that factories discharge after making products? Or the water that runs off of roads and farmlands after a big storm? Water and liquid waste treatment plant and system operators run the amazing water treatment plants that remove pollutants and other harmful materials from waste water, so that it can be safely returned to the environment. These operators provide essential services that everyone in the community depends on every day to keep our water supply safe and clean.			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.

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