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Project Summary

Difficulty	6
Time required	Average (about one week)
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.

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Abstract

Objective

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

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.

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.

Terms, Concepts and Questions to Start Background Research

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:
  
  • Van Hoeck, Baker and Tokuhisa, 2004. "What is a zone of inhibition on an agar plate?" Ask A Scientist, Molecular Biology Archive, University of Chicago and Argonne National Laboratories, Newton Bulletin Board System [accessed September 11, 2006] http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm.
  • Rollins, D.M. and S.W. Joseph, 2000. "Antibiotic Disk Susceptibilities," Department of Cell Biology and Molecular Genetics, University of Maryland, College Park [accessed September 11, 2006] http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• at least 6 nutrient agar plates:
  • 3 plates will serve as controls, with no disinfectants,
  • 3 plates will serve as test plates, with disinfectant disks.
• live E. coli culture (commonly available in labs; can also be purchased from online suppliers),
• sterile swabs,
• sterile tube with 10 ml sterile water,
• filter paper,
• hole punch,
• forceps,
• permanent marker,
• disinfectants (up to six), 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).

Experimental Procedure

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. Prepare sterile filter disks by using a hole punch to make small circular disks from filter paper. You can use pencil or permanent marker to label each disk with a code for the disinfectant to be used for that disk (up to six). Keep track of the codes in your lab notebook. Wrap 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 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 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. Prepare a dilute solution of bacteria for innoculating the plates.
   1. Wipe a sterile swab across the surface of an existing plate (prepare from your E. coli culture 24 hours in advance).
   2. Using proper sterile technique, open the tube of sterile water and swirl the swab in the water.
   3. Cap the tube.
   4. Properly dispose of the contaminated swab. Agitate the tube before using.
5. To innoculate a plate, dip a sterile swab in the dilute bacterial solution, using proper sterile technique. 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.
6. 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.
7. 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.
8. Incubate all of the plates, inverted, (agar on top) overnight. 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 diameter, 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 around each disk. Keeping the lid of the plate in place, use a ruler to measure the diameter of the clear area in millimeters. 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

Safe Disposal of Plates

At the conclusion of the experiment, all plates should be disinfected for safe disposal.

1. The best way to dispose of bacterial cultures is to pressure-sterilize (autoclave) them in a heat-stable biohazard bag.
2. If autoclaves or pressure cookers are not available, an alternative is to bleach the plates.
   1. Wear proper safety equipment (gloves, lab coat, eye protection) when working with the bleach solution; it is corrosive.
   2. Saturate the plates with a 20% household bleach solution (in other words, one part bleach and four parts water).
   3. Allow the plates to soak overnight in the bleach solution before disposing of them.
   4. Please note that the bleach solution is corrosive and needs to be thoroughly rinsed afterwards.

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.
• 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?

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

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.

Last edit date: 2007-03-22 22:00:00

Career Focus

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

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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