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Chemical Methods of Control
Classroom Activity

Access Excellence Classic Collection


Adapted from T. Johnson and C. Case. Laboratory Experiments in Microbiology, Brief Edition, 4th ed., Redwood City, CA: Benjamin/Cummings Publishing Co., 1995.

Objectives:

The student will be able to:
  1. Define the following terms: disinfectant and antiseptic.
  2. Evaluate the relative effectiveness of various chemical substances as antimicrobial agents.

Background:

A wide variety of chemicals called antimicrobial agents are available for controlling the growth of microbes. For example:
  • Chemotherapeutic agents, including antibiotics, are used internally.
  • Disinfectants are chemical agents used on inanimate objects to lower the level of microbes present on the object.
  • Antiseptics are chemicals used on living tissue to decrease the number of microbes present in that tissue.

Disinfectants and antiseptics affect bacteria in many ways. Those that result in bacterial death are called bactericidal agents. Those causing temporary inhibition of growth are bacteriostatic agents.

No single antimicrobial agent is most effective for use in all situations - different situations may call for different agents. A number of factors affect selection of the best agent for any given situation - Antimicrobial agents must be selected with specific organisms and environmental conditions in mind. Additional variables to consider in the selection of an antimicrobial agent include pH, solubility, toxicity, organic material present, and cost.

Once an agent has been selected, it is important to evaluate it's effectiveness. In evaluating the effectiveness of antimicrobial agents, the concentration, length of contact, and whether it is lethal (-cidal) or inhibiting (-static) at that concentration and exposure are the important criteria.

One method of measuring the effectiveness of a chemical agent is to determine its zone of inhibition. In the agar diffusion method, one species of bacteria is uniformly swabbed onto a nutrient agar plate. Chemicals are placed on paper disks. These discs are added to the surface of the agar. During incubation, the chemical diffuses from the disk containing the agent into the surrounding agar. An effective agent will inhibit bacterial growth, and measurements can be made to quantify the size of the zones of inhibition around the disks. The relative effectiveness of a compound is determined by comparing the diameter of the zone of inhibition with values in a standard table.

The agar diffusion test is not used to determined whether a chemical is bactericidal (kills bacteria) or bacteriostatic (inhibits bacteria) - instead this characteristic is determined by the dilution method. In this method the bacterium of interest is placed in a tube containing the chemical which is being tested. The bacterium is then added (subcultured) onto a nutrient agar plate. If the bacterium grows on the nutrient agar the chemical is bacteriostatic; if not, it was killed by the chemical which is then termed "bactericidal."

Materials:

  • Petri plates containing nutrient agar (2)
  • Sterile cotton swab
  • Forceps
  • Ruler
  • Disks (use a hole punch on filter paper)
  • Test substances: Garlic powder, chemical agents such as bathroom cleaner, floor cleaner, mouthwash, lens cleaner, acne cleaner. Bring your own.
  • Staphylococcus epidermis (Gram-positive)
  • Escherichia coli (Gram-negative)

Table 1: Interpretation of Inhibition Zones of Test Cultures

Diameter of Zones of
Inhibition (mm)
Resistant 10 or less
Intermediate 11-15
Susceptible 16 or more

Procedure:

  1. With a wax marking pencil or magic marker, divide a nutrient agar plate into six sections. Label the sections 1 through 6 on the bottom of the plate.

  2. Aseptically swab the assigned culture onto the appropriate plate. Swab in three directions to ensure complete plate coverage. Why is complete coverage (link s1) essential? Let stand at least 5 minutes.

  3. Obtain a disk impregnated with a disinfectant and place it on the surface of the agar. With forceps (link s 2), gently tap the disk to ensure better contact with the agar. Repeat, placing six disks of different disinfectants the same distance apart on the Petri plate. Record the names of the disinfectants so that you know which plate contains each disinfectant.

  4. Add a small amount of garlic powder or a penny (link s 3) to an unused area.

  5. Incubate inverted(agar on top) at 35 degrees C until the next period. Working with the lid of the Petri plate in place, use a ruler to measure the zones of inhibition in millimeters. Record the zone size and, based on the values in Table 1(above), indicate whether the organism is sensitive, intermediate, or resistant to the effects of the tested bacteria/disinfectant pair. Observe the results (link s 4)of students using the other bacteria and other disinfectants.

Results:

Agar diffusion method. Record your results and those of another student group using the other bacterium.

ChemicalZone of inhibition
E.coliS.epidermidis

Questions:

  1. Which chemical was not effective against a gram-positive bacterium? Against a gram-negative bacterium? How does this influence where you use these chemicals?
  2. Is this a fair measure of how a chemical will perform on an environmental surface? Briefly explain why or why not.
  3. How could you determine whether the bacteria in a zone of inhibition were killed or just inhibited?

Additional Activities:

  1. Compare the antimicrobial effectiveness of competing products.
  2. Use different dilutions of the test substance.
  3. 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.

Classroom Discussion:

  1. Why are there many types of antibiotics?
  2. Have students research antibiotic resistance. Was antibiotic resistance demonstrated in the experiments above?


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