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1.40: Bacterial Susceptibility to Antibiotics (Kirby-Bauer Test)

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    Learning Objectives
    • Define "antibiotic" and tell where these chemicals come from in nature (for non-synthetic or non-semisynthetic).
    • Differentiate between broad spectrum antibiotics and narrow spectrum antibiotics.
    • Tell that use of broad spectrum antibiotics contributes to increased antibiotic resistance in bacteria.
    • Tell the purpose of the Kirby-Bauer test.
    • Explain how the Kirby-Bauer test works including how diffusion of the antibiotics is important in the creation zones of inhibition.
    • Define "zone of inhibition."
    • Successfully conduct a Kirby-Bauer test (modified) and interpret the results.
    • Explain why the Kirby-Bauer test is important for reducing antibiotic resistance.
    • Describe why antibiotic resistance is a threat for successful treatment of bacterial infections.


    Introduction to Antibiotics

    Antibiotics are chemicals produced by some bacteria and fungi that, in small quantities, can inhibit the growth of bacteria. Much of the success we have achieved in treating infections since World War II is due to the discovery of antibiotics. Utilizing the information gleaned from studying these natural chemicals, scientists have artificially synthesized other useful antimicrobial chemicals in the laboratory. Sometimes these antimicrobials are completely synthesized in the lab (synthetic) and sometimes they are partly produced in nature and partly synthesized in the lab (semisynthetic).

    Many microbes can produce antibiotics, but four genera produce most of the antibiotics used for treating human and animal infections. Bacillus and Streptomyces are bacteria. Penicillium and Cephalosporium are fungi. It is a constant challenge to develop new antibiotics to replace those antibiotics for which microbes have developed resistance.

    The range of bacteria killed by an antibiotic determines its “spectrum of activity”. Antibiotics that are only effective against Gram-positive or only effective against Gram-negative bacteria have a narrow spectrum of activity. Antibiotics that are effective against many different types of bacteria are called broad spectrum antibiotics. Broad spectrum antibiotics are probably contributing to the escalating drug resistance we are seeing in microorganisms. Broad spectrum antibiotics often wipe out a person’s normal microbiome as well as the pathogen they are intended to kill, resulting in superinfections from organisms such as Candida albicans and Clostridium difficile that grow out of control when they do not have to compete with microbes in the normal microbiomes.

    Empiric therapy takes place when an antimicrobial agent is given to the patient without performing a culture or other diagnostic test to determine the specific cause of the disease. Empiric therapy is prescribed in instances where the causative pathogen is likely and where diagnostic tests will not change the treatment. The selection of which drug to use is based solely on experience, observation and relevant clinical information including current resistance patterns in suspected pathogens. These antibiotics are typically broad-spectrum, in that they treat a wide variety of possible microorganisms. Examples of this include antibiotics prescribed for strep throat, pneumonia, urinary tract infections, and suspected bacterial meningitis in newborns aged 0 to 6 months.

    Physicians are beginning to target infections with narrow spectrum antibiotics, or synergistically treat infections with small doses of multiple antibiotics to try to prevent antibiotic resistance.

    The laboratory can aid the physician in selecting which antimicrobial agent is likely to kill the pathogen that is causing an infection in a patient. There are several methods that are used by clinical microbiologists in this determination, including the Kirby-Bauer Test.


    Kirby-Bauer Test

    The results of the Kirby-Bauer Test provide an accurate prediction of which antibiotics are likely to be effective against the pathogen. Because the Kirby-Bauer test is relatively simple to perform and is inexpensive, it has been extensively used in medical practice. The results are reported as S (sensitive), I (intermediate), and R (resistant) to an antibiotic. A sensitive result indicates the bacteria will die when it is exposed to the antibiotic. An intermediate result indicates the antibiotic must be used in combination with another antibiotic to clear the infection. A resistant result indicates the antibiotic does not kill the bacteria.

    streaking a petri plate with bacteria and adding antibiotic discs produces circular regions where there is no growth surrounding the antibiotic discs called zones of inhibition

    Figure 1: A summary diagram showing how the Kirby-Bauer approach works. A petri plate with nutrient agar is spread with the bacteria being tested. Antibiotic disks are placed on the agar. After incubation, zones on inhibition will occur surrounding the antibiotic disks based on the bacteria's sensitivity to the antibiotics tested. The zone of inhibition is a region where no bacterial growth occurred since it was inhibited/killed by the concentration of antibiotic present on the petri plate at that location (based on the diffusion of the antibiotic away from the antibiotic disk).


    Petri plates with nutrient agar are covered with the bacteria that is being tested for its antibiotic sensitivity. After spreading the bacteria over the entire surface of the petri plates, small disks containing different antibiotics are placed at a distance from each other (or alone) on the petri plate. Each antibiotic will diffuse outward from the antibiotic disk creating a concentration gradient of the antibiotic in a circle surrounding the disk. Closest to the antibiotic disk will be the highest concentration of antibiotic. Further from the antibiotic disk will be the lowest concentration of antibiotic. Based on how sensitive the bacterial species/strain is to each antibiotic, bacterial will begin to grow at one of the antibiotic concentrations. Bacteria that can grow at higher concentrations of an antibiotic (closer to the disk) will have more resistance to that antibiotic. Bacteria that can only grow at lower concentrations of an antibiotic (further from the disk) will be more susceptible to that antibiotic. As a result, the size of the zone of inhibition (the region of the petri plate surrounding the antibiotic disk that does not have bacterial growth) will determine if the bacterial species/strain is sensitive, intermediate, or resistance to that antibiotic.


    zones of inhibition are concentration gradients of the antibiotic with the highest concentration near the antibiotic disc and the lowest antibiotic concentration further away from the disc

    Figure 2: A closer look at antibiotic diffusion and how this produces zones of inhibition. Closest to the antibiotic disk is the highest antibiotic concentration and the antibiotic diffuses outward to produce a concentration gradient of antibiotic. The further from the antibiotic disk, the lower the antibiotic concentration. This approach enables a quick way of seeing a spectrum of antibiotic concentrations and how this impacts inhibition of the bacterial species/strain being tested.


    The Kirby-Bauer test, known as agar disk diffusion, must be strictly regulated for the results to be interpreted correctly. Such characteristics as the stability of the antibiotic, the rate of diffusion of the antibiotic, the bacteria being tested, the pH of the culture medium, the depth of the culture medium, the inoculum density, the incubation time, the incubation temperature, and the concentration of the antibiotic can affect the results. Nevertheless, when the Kirby-Bauer test is performed under standardized conditions (on Mueller-Hinton agar inoculated with a pure culture of microbes that is the correct turbidity to match McFarland 0.5 standard, etc.) and the results are interpreted according to the Interpretative Zone Standards published by the National Committee for Clinical Laboratory Standards. In this laboratory, this is a simplified version of the Kirby-Bauer Test in lab that is not standardized, but will allow you to learn the general principles involved in this procedure.


    Laboratory Instructions


    Kirby-Bauer Test for Antibiotic Sensitivity (Modified for Simplicity)

    1. Label 3 TSA plates with your group name and "Kirby-Bauer."
    2. Dip a sterile cotton swab into a Staphylococcus aureus TSB culture and completely coat the surface of the TSA plate with S. aureus. Cotton swabs should go into an antimicrobial solution and be left there for at least 10 minutes before disposal.
    3. Repeat step 2 for all the TSA plates.
    4. Allow the plates to dry for 5 – 10 minutes.
    5. Gently place one penicillin G disk (disk is labeled as P) in the center of one of the petri plates. DO NOT press the disk into the agar and do not move the disk once placed on the agar.
    6. Repeat step 5. for an erythromycin disk (disk is labeled as E) on a different petri plate.
    7. On the third petri plate, place a streptomycin disk (disk is labeled as S) centrally on one side of the petri plate and a tetracycline disk (labeled as T or TE) centrally on the other side of the same petri plate making sure they are spread out from each other.
    8. DO NOT INVERT THE PLATES! Place the petri plates in the incubator.
    9. After given time for growth (24-48 hours), measure the zones of inhibition in mm and record results in the results table.
    10. Identify which of the size ranges that each zone of inhibition falls into in the interpretation table. This will determine if this bacterial strain is susceptible, intermediate, or resistant to each antibiotic.


    measurement of the diameter of zone of inhibition on a petri plate

    Figure 3: Example of how to measure the diameter of a zone of inhibition in mm. The example shown here has a zone of inhibition with a diameter of 32 mm.


    Results & Questions


    antibiotic disk abbreviation

    antibiotic name

    antibiotic disk concentration

    strain is resistant for this size of zone of inhibition (mm)

    strain is intermediate for this size of zone of inhibition (mm)

    strain is susceptible for this size of zone of inhibition (mm)



    15 µg

    13 or less

    14 – 22

    23 or more


    penicillin G

    10 units

    28 or less


    29 or more



    10 µg

    6 or less

    7 – 9

    10 or more

    T (TE)


    30 µg

    14 or less

    15 – 18

    19 or more

    antibiotic disk

    diameter of the zone of inhibition (mm)

    this S. aureus strain is... (resistant, intermediate or susceptible)... to this antibiotic

    E (erythromycin)


    P (penicillin G)


    S (streptomycin)


    T (TE) (tetracycline)

    1. Complete the table above using measurements of the diameter of the zones of inhibition and the interpretation table above to determine if S. aureus is resistant, intermediate, or susceptible to each antibiotic.
    2. If you had a patient with an infection of this strain of S. aureus, which antibiotic(s) might be a good choice for treatment? Explain your answer.
    3. Define "zone of inhibition."
    4. Explain how the size of the zone of inhibition relates to the concentration of the antibiotic.
    5. Explain how the size of the zone of inhibition relates to the susceptibility or resistance to an antibiotic.
    6. What is "antibiotic resistance?"
    7. Why is using this Kirby-Bauer approach helpful to prevent bacterial strains from becoming resistant to antibiotics?
    8. True or False. It is easy for scientists to develop new antibiotics.
    9. What are the treatment options for a bacterial infection where the bacterial species is resistant to all types of antibiotics?
    10. Why is it important to take steps to prevent bacterial strains from becoming antibiotic resistant?



    This page titled 1.40: Bacterial Susceptibility to Antibiotics (Kirby-Bauer Test) is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.

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