Home Store Project Ideas Project Guide Ask An Expert Blog Careers Teachers Parents Students

The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition

Difficulty
Time Required Average (6-10 days)
Prerequisites To do this project, you will need access to a laboratory with facilities for culturing bacteria. You should be familiar with sterile technique and proper handling of bacterial cultures.
Material Availability Specialty items
Cost Average ($50 - $100)
Safety Standard precautions for handling bacterial cultures and bleach.

Abstract

Have you heard that garlic powder is supposed to inhibit the growth of bacteria? Which do you think would make a better disinfectant: a solution of garlic powder or a solution of bleach? This project shows you a straightforward way to compare the effectiveness of different disinfectants (or other antimicrobial agents), by measuring zones of inhibition on a culture plate.

Objective

The goal of this project is to measure the effectiveness of different antimicrobial agents by measuring zones of inhibition on bacterial culture plates.

Credits

Andrew Olson, Ph.D., Science Buddies

Sources

This project is based on:

  • Johnson, T. and C. Case, 1995. "Chemical Methods of Control," adapted from Laboratory Experiments in Microbiology, Brief Edition, 4th ed. Redwood City, CA: Benjamin/Cummings Publishing Co., available online from The National Health Museum, Access Excellence Activities Exchange [accessed September 11, 2006] http://www.accessexcellence.org/AE/AEC/CC/chance_activity.html.

Cite This Page

MLA Style

Science Buddies Staff. "The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition" Science Buddies. Science Buddies, 6 Dec. 2014. Web. 20 Dec. 2014 <http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_p014.shtml?from=Blog>

APA Style

Science Buddies Staff. (2014, December 6). The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition. Retrieved December 20, 2014 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_p014.shtml?from=Blog

Share your story with Science Buddies!

I did this project I Did This Project! Please log in and let us know how things went.


Last edit date: 2014-12-06

Introduction

Antimicrobial agents are chemicals that are used against bacteria. There are many such agents available. Because there are many different situations where bacterial control is important, no antimicrobial agent is effective in all situations. For example, you wouldn't use the same compound to fight an ear infection as you would use to sterilize surfaces in an operating room. The situations are completely different. In one case, you are trying to assist the body to fight off an internal infection, and in the other case, you are trying to eliminate bacteria from inanimate surfaces.

There are many additional factors that you would have to consider in order to choose an appropriate antimicrobial agent for a given situation. For example, are the chemical properties of the agent (e.g., pH and solubility) appropriate for the situation? You would want to know whether the compound is toxic—to humans, other animals, plants, or beneficial bacteria. Finally, you would definitely want to know that the compound is effective against the organism(s) you are trying to eliminate.

This project shows you one method of measuring the effectiveness of an antimicrobial agent against bacteria grown in culture. This is called the Kirby-Bauer disk-diffusion method, and here is how it works. The bacteria of interest is swabbed uniformly across a culture plate. Then a filter-paper disk, impregnated with the compound to be tested, is placed on the surface of the agar. The compound diffuses out from the filter paper into the agar. The concentration of the compound will be higher next to the disk, and will decrease gradually as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow wherever the concentration in the agar is greater than or equal to that effective concentration. This region is called the "zone of inhibition." Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound. Figure 1, below, illustrates the idea.

zones of inhibition on a bacterial culture plate
Figure 1. The illustration above shows zones of inhibition around filter paper disks saturated with anti-microbial compounds. The diameter of the zone of inhibition is a measure of the effectiveness of an anti-microbial compound (Rollins and Joseph, 2000).

You can use this method to compare the effectiveness of different disinfectants or different antibiotics against a strain of bacteria. Since this method depends on diffusion of the compound, it is important to keep several factors constant when you make your comparisons, including:

  • the size of the filter disks,
  • the temperature of incubation,
  • the composition and thickness of the agar, and
  • the uniformity of bacterial plating.

With careful attention to making your conditions consistent, this method will produce reliable results for comparing antimicrobial effectiveness.

Terms and Concepts

To do this project, you should do research that enables you to understand the following terms and concepts:

  • colony,
  • zone of inhibition,
  • disinfectant,
  • antibiotic,
  • antiseptic.

Bibliography

This project is based on:

  • Johnson, T. and C. Case, 1995. "Chemical Methods of Control," adapted from Laboratory Experiments in Microbiology, Brief Edition, 4th ed. Redwood City, CA: Benjamin/Cummings Publishing Co., available online from The National Health Museum, Access Excellence Activities Exchange [accessed September 11, 2006] http://www.accessexcellence.org/AE/AEC/CC/chance_activity.html.
  • For more background information on zones of inhibition, see these references:

News Feed on This Topic

 
, ,
Reading level:
Note: A computerized matching algorithm suggests the above articles. It's not as smart as you are, and it may occasionally give humorous, ridiculous, or even annoying results! Learn more about the News Feed

Materials and Equipment

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

These items can be purchased from Carolina Biological Supply Company, a Science Buddies Approved Supplier:

  • Sterile disks (3 per disinfectant tested). Alternatively, disks may be made using a hole punch and filter paper, but they will need to be sterilized in the oven, as described in the Procedure.
  • Nutrient agar plates (6):
    • 3 plates will serve as controls, with no disinfectants,
    • 3 plates will serve as test plates, with disinfectant disks.
  • Sterile cotton-tipped applicator swabs. Alternatively, cotton-tipped swabs from a new, unopened box may be used.
  • Live E. coli, strain K-12
  • Forceps. Alternatively, tweezers may be used.
  • 70% ethanol or isopropyl rubbing alcohol, for using sterile technique. Alternatively, this can be purchased in the disinfectant section of a grocery store or drug store. Note: If you are ordering this chemical through Carolina Biological Supply Company, the chemical must be ordered by a teacher and shipped to a school or business address, so plan accordingly.
  • Disposable gloves. Alternatively, these can be purchased at a local drug store or pharmacy. If you are allergic to latex, use vinyl or polyethylene gloves.

You will also need to gather these items

  • Permanent marker
  • Optional: Pencil
  • Optional: Aluminum foil and oven
  • Timer or clock
  • Disinfectants (up to 6). Here are some ideas for different compounds to test:
    • Solution of garlic powder
    • Liquid bathroom cleaner
    • Liquid floor cleaner (e.g., one containing pine oil)
    • Mouthwash
    • Contact lens cleaner
    • Anti-acne product
    • Household bleach (sodium hypochlorite)
  • Optional: 37°C incubator for bacterial culture plates
  • Ruler, metric
  • 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 does participate in affiliate programs with Amazon.comsciencebuddies, Carolina Biological, and AquaPhoenix Education. Proceeds from the affiliate programs help support Science Buddies, a 501( c ) 3 public charity. 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.

Share your story with Science Buddies!

I did this project I Did This Project! Please log in and let us know how things went.

Experimental Procedure

Working with Biological Agents

For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. Many science fairs follow Intel® International Science and Engineering Fair (ISEF) regulations. For more information, visit these Science Buddies pages: Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee. You can also visit the webpage ISEF Rules & Guidelines directly.

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 Disk Diffusion Test

For this experiment, it is important to innoculate the plate with a uniform distribution of bacterial colonies, and to use the exact same procedure for each plate. Here are the steps for innoculating the control and test plates.

  1. You can use a pencil or permanent marker to label each sterile disk with a code for the disinfectant to be used for that disk (up to six). If you label the disks, keep track of the codes in your lab notebook, wrap the disks in aluminum foil, and sterilize in a 300° oven for 30 minutes.
  2. Use a permanent marker to mark the bottoms of the three nutrient agar plates that will be your test plates with as many sections as you have disinfectants (up to six). The sections should all be equal in size. Number the sections sequentially.
  3. Label the three nutrient agar plates that will be your control plates. The purpose of these plates is to show that the bacteria consistently grow uniformly over the plate in the absence of disinfectant disks—confirming that your innoculation technique is consistent, and that the plates support uniform bacterial growth.
  4. Using proper sterile technique, inoculate each plate uniformly. Dip a sterile cotton-tipped applicator swab in the K-12 E. coli bacterial solution. Gently wipe the swab over the surface of the plate, swabbing in three directions (120° apart) to insure complete coverage of the plate. Cover the plate and wait at least five minutes for the plate to dry.
  5. Hold a single sterile disk by the edge with sterile forceps and dip it into the disinfectant solution to be tested (make sure it matches with the label on the disk). Touch the disk against the side of the container to drain off excess liquid.
  6. Use sterile forceps to place a single disinfectant disk in the center of each of the marked sections on your test plates. Use the forceps to gently press each disk against the agar surface to insure good contact. Remember to use the exact same technique for each disk—consistency is very important for this experiment. Take notes in your lab notebook to keep track of which disinfectant is tested in each numbered section.
  7. Incubate all of the plates, inverted (agar on top), overnight at 37°C. Use a longer incubation time if necessary (for example, for incubation at lower temperature).

Measuring Zones of Inhibition

  1. After overnight incubation, examine your plates (keep them covered at all times).
    1. The control plates should show uniform colonies over the entire surface of the plate. If the distribution is highly uneven, you will need to improve your innoculation technique and repeat the experiment.
    2. If your disinfectants are effective at the concentrations you tested, you should see zones of inhibition around the disinfectant disks. The clear zones around each disk should have a uniform width, since diffusion of the compounds through the agar should be uniform in every direction. If this is not the case, suspect either your impregnation technique, or poor contact of the filter paper with the agar.
  2. Measure the diameter of the zone of inhibition for each disk. Keeping the lid of the plate in place, use a ruler to measure the diameter of the disk plus the surrounding clear area in millimeters (mm).
    1. Include the diameter of the disk in your measurements. For example, if your disk has a diameter of 6 mm and the clear area has a width of 3 mm beyond the disk, the diameter of the zone of inhibition that you should measure and record would be 12 mm (6 mm + 3 mm + 3 mm). This is the standard way that zones of inhibition are measured.
    2. You will get three separate measurements for each disinfectant, one from each of the three test plates.
  3. Are the diameters consistent across all three plates? Calculate the average and the standard deviation of the diameter of the zone of inhibition for each disinfectant.
  4. Use the values from Table 1 (below) to evaluate the bacterial response to each compound (Johnson and Case, 1995).

      Diameter of zone of inhibition (mm)
    Resistant 10 or less
    Intermediate 11–15
    Susceptible 16 or more

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. Additionally, many science fairs follow ISEF Rules & Guidelines, which have specific guidelines on how bacteria and other microorganisms should be handled and disposed of.

  • 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.

Share your story with Science Buddies!

I did this project I Did This Project! Please log in and let us know how things went.


Variations

  • Increase the number of bacterial strains to test. For example, you could also test disinfectants on a gram-positive bacterium, such as Stapylococcus epidermis. Is S. epidermis susceptible to the same disinfectants as E. coli?
  • Test bacterial susceptibility to antibiotics. You can purchase pre-made antibiotic disks to use for this experiment from online suppliers. A search for "antibiotic disks" should turn up several alternatives. Do background research on each antibiotic to learn about its mechanism of action for killing bacteria. For this experiment, it is a good idea to double the number of plates and use two strains of bacteria, one gram-positive and one gram-negative.
  • In some parts of the world traditional medicine, also called indigenous or folk medicine, uses seaweed to keep wounds clean. Scientists, as described in this research article, are also interested in testing the medicinal value of seaweed and smaller algae. Try adapting the Procedure of this project to look at whether or not different seaweeds and algaes have antibacterial properties.
  • Use different dilutions of the test substance. For example, you could try a series of 2-fold dilutions of each test compound. Do you see a decrease in the size of the zone of inhibition as concentration is decreased? At what concentration does each disinfectant tested become ineffective?
  • Test antiseptics against a bacterium isolated from your body. Scrape your teeth with a toothpick and add to a nutrient agar plate. Take a swab and swab the teeth scrapings onto the surface. Add the discs with chemical substances such as mouth wash (Johnson and Case, 1995).
  • For a more advanced experiment to investigate development of bacterial resistance to disinfectants, see the Science Buddies project: Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?

Share your story with Science Buddies!

I did this project I Did This Project! Please log in and let us know how things went.

Ask an Expert

The Ask an Expert Forum is intended to be a place where students can go to find answers to science questions that they have been unable to find using other resources. If you have specific questions about your science fair project or science fair, our team of volunteer scientists can help. Our Experts won't do the work for you, but they will make suggestions, offer guidance, and help you troubleshoot.

Ask an Expert

Related Links

If you like this project, you might enjoy exploring these related careers:

female microbiologist looking in microscope

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. Read more
clinical technician testing blood types

Medical & Clinical Laboratory Technician

Doctors need information to decide if a person is healthy or sick, if a baby's earache is bacterial or viral, or if the man next door needs medication to lower his cholesterol and prevent a heart attack. The information often comes in the form of results from lab tests. Medical and clinical laboratory technicians are the people who perform these routine medical laboratory tests, giving the doctors the information needed to diagnose, treat, and prevent disease. Read more
biological technician working at laboratory bench

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 biotechnology advances is often the work of skilled biological technicians. Read more

News Feed on This Topic

 
, ,
Reading level:
Note: A computerized matching algorithm suggests the above articles. It's not as smart as you are, and it may occasionally give humorous, ridiculous, or even annoying results! Learn more about the News Feed

Looking for more science fun?

Try one of our science activities for quick, anytime science explorations. The perfect thing to liven up a rainy day, school vacation, or moment of boredom.

Find an Activity