The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition
|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||A kit for this project is available from our partner Home Science Tools.|
|Cost||Average ($50 - $100)|
|Safety||Follow the general safety precautions for handling microorganisms outlined at the end of the Procedure for this science project.|
AbstractHave 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.
The goal of this project is to measure the effectiveness of different antimicrobial agents by measuring zones of inhibition on bacterial culture plates.
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. Retrieved September 11, 2006, from http://www.accessexcellence.org/AE/AEC/CC/chance_activity.html.
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Last edit date: 2018-12-12
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.
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:
- Zone of inhibition
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. Retrieved December 10, 2018, from http://www.accessexcellence.org/AE/AEC/CC/chance_activity.html.
- For more background information on zones of inhibition, see:
- Rollins, D.M. and S.W. Joseph, 2000. "Antibiotic Disk Susceptibilities," Department of Cell Biology and Molecular Genetics, University of Maryland, College Park. Retrieved December 10, 2018, from http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm.
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- Neutralizing Bacteria Kit, available from our partner
Home Science Tools. Needed from the kit:
- Nutrient agar plates (12)
- E. coli culture, freeze dried and ready to reconstitute
- Sterile disks (48)
- Sterile cotton swabs (6)
- Nitrile gloves
- You will also need to gather these items, not included in the kit:
- Permanent marker
- Stove or other way of heating water to boiling
- Water, 1 cup
- 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)
- Contact lens cleaner
- Anti-acne product
- Household bleach (sodium hypochlorite)
- Optional: 37°C incubator for bacterial culture plates
- Ruler, metric
- Lab notebook
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The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition
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 inoculate the plate with a uniform distribution of bacterial colonies, and to use the exact same procedure for each plate. Here are the steps for inoculating the control and test plates.
- Use a permanent marker to divide the bottom of 12 nutrient agar plates into four quadrants each. In one quadrant of each plate write "C" for control. Write the name of a different disinfectant in each remaining quadrant. In summary:
- Every plate should have one control quadrant and 3 different disinfectant quadrants.
- Each disinfectant should be represented on 6 different plates.
- Follow the directions in the kit to reconstitute the dried E. coli. Let the reconstituted E. coli sit at room temperature throughout the next step.
- Sterilize a cup of water by boiling it on the stove top for 5 minutes. Cover and wait for it to cool to room temperature. Allowing the water to cool is critical; if it is too hot then it will kill the bacteria in the next step.
- Using proper sterile technique, inoculate each plate uniformly. While wearing gloves, gently shake the container of reconstituted E. coli so that it is uniformly mixed. Put two drops of the E. coli mixture on a plate. Dip a fresh (unused) sterile cotton swab into the sterile water. Use the cotton swab to wipe the drops of bacteria around the entire surface of the plate. Cover the plate and wait at least five minutes for the plate to dry. A few additional tips:
- The sterile water on the cotton swab makes it easier to spread the bacteria uniformly.
- Make sure to use a fresh swab for each plate. Do not dip a used swab back in to the sterile water.
- Hold a single sterile disk by the edge with sterile forceps and dip it into the disinfectant solution to be tested. Touch the disk against the side of the container to drain off excess liquid.
- Use sterile forceps to place a single disinfectant disk in the center of each of the quadrants 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. Make sure to carefully match the label on the plate with the right disinfectant.
- Place a plain (not dipped in any disinfectant) sterile disk in each control quadrant.
- Incubate all of the plates, inverted (lid on the bottom and 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
- After overnight incubation, examine your plates (keep them covered at all times).
- The control quadrants should show uniform colonies over the entire surface of the plate. If the distribution is highly uneven, you will need to improve your inoculation technique and repeat the experiment.
- 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.
- 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).
- 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.
- You will get six separate measurements for each disinfectant, one from each of the three test plates.
- 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.
- 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|
|Susceptible||16 or more|
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.
- 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.
If you like this project, you might enjoy exploring these related careers:
- 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 algae 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?
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