Interpret results of Kirby-Bauer disk diffusion susceptibility tests

Differentiate between the Kirby-Bauer disk diffusion susceptibility test  and the E-test

Interpret results of E-tests

Interpret the result of a beta-lactamase test


Describe and compare the effectiveness of physical and chemical methods of microbial control

Identify structural characteristics of the major groups of microorganism

Compare and contrast prokaryotic cell and eukaryotic cells

Compare and contrast the physiology and biochemistry of the various groups of microorganisms

Apply various laboratory techniques to identify types of microorganisms


Mueller-Hinton agar plates with confluent lawns of:

  • Escherichia coli (Gram-negative)
  • Pseudomonas aeruginosa (Gram-negative)
  • Staphylococcus aureus (Gram-positive)

Antibiotic disks for Kirby-Bauer Disk Diffusion Susceptibility Test:

  • Zithromax (brand name) or Azithromycin (generic)
  • Augmentin (brand name) or Amoxicillin and Clavulanic Acid (generic)
  • Cipro (brand name) or Ciprofloxacin (generic)
  • Bactrim (brand name) or Sulfamethoxazole and Trimethoprim (generic)
  • Neosporin (brand name, topical) Poly-Rx (brand name, systemic) or Polymyxin B (generic)


  • Cipro (brand name) or Ciprofloxacin (generic)

Metric Ruler

Class Demonstration:

Beta-Lactamase Test

Antibiotics are chemicals produced by some bacteria and fungi that, in small quantities, 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 naturally produced chemicals, scientists have artificially synthesized other useful antibiotics in the laboratory. Sometimes these antibiotics 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.
How do antibiotics inhibit the growth of bacteria? There are five targets of antibiotics:

Bacterial Cell Wall Synthesis Blocks peptidoglycan synthesis, osmotic forces cause bacteria to shrivel or burst
Disrupt Membranes Disorganize the structure or inhibit the function of membranes
Nucleic Acid Synthesis Block synthesis of DNA or RNA or bind to DNA or RNA so they cannot be replicated, transcribed, or translated
Protein synthesis Target bacterial 70s ribosome
Metabolic Pathways Act as a competitive inhibitor and occupy the active site of an enzyme so the substrate can’t bind

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.

How do physicians choose the correct antibiotic to treat an infection? Empiric therapy takes place when an antibiotic 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.

Laboratory tests can aid the physician in selecting which antibiotic and dosage is likely to kill the pathogen that is causing an infection in a patient. There are several methods that are used including the Kirby-Bauer disk diffusion susceptibility test and the E-test used to determine the minimum inhibitory concentration (MIC).

Because the Kirby-Bauer test is simple to perform and is relatively inexpensive, it has been extensively used in medical practice. The Kirby-Bauer disk diffusion susceptibility test determines the sensitivity or resistance of pathogenic bacteria to various antibiotics in order to assist physicians in selecting antibiotics for their patients. The pathogenic organism is grown on Mueller-Hinton agar in the presence of antibiotic impregnated filter paper disks. The antibiotic will diffuse out of the disk and into the media. The presence or absence of growth around the disks is an indirect measure of the ability of that antibiotic to inhibit bacterial growth.

Kirby-Bauer test 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 antimicrobial. An intermediate result indicates the antimicrobial must be used in combination with another antimicrobial to clear the infection. A resistant result indicates the antimicrobial does not kill the bacteria.

Kirby-Bauer Test Figure
An effective antibiotic will produce a large zone of inhibition (disk C), while an ineffective antibiotic may not affect bacterial growth at all (disk A). Antibiotics to which a bacterial isolate is partially susceptible will produce an intermediate size zone of inhibition (disk B).
Kirby-Bauer Disk Diffusion Susceptibility Test
Kirby-Bauer Disk Diffusion Susceptibility Test

The epsilometer or E-Test combines a disk diffusion susceptibility pattern with the determination of Minimum Inhibitory Concentration (MIC). The MIC test determines the lowest concentration of an antibiotic at which bacterial growth is completely inhibited therefore MIC determines the dosage of the antibiotic that should be effective in controlling the infection in the patient. An antibiotic gradient is present on the E-Test plastic strip.

E-Test Strip
E- Test strip

When the E- test strip is applied to an inoculated Muller-Hinton agar plate, there is an immediate release of the antibiotic and the establishment of an antibiotic concentration gradient on the agar. After incubation, bacterial growth becomes visible on the plate, and a symmetrical inhibition ellipse along the strip is seen. The MIC value in ug/mL is read at the point where ellipse intersects the scale of the E-test strip.

Determining MIC using and E- Test
Determining MIC using an E-Test

The following antibiotics were tested:

Zithromax (brand name)

Azithromycin (generic)

Semi-synthetic Protein synthesis Ear, Skin, Eye, Respiratory, Sinus, and Sexually Transmitted Infections
Augmentin (brand name)

Amoxicillin and Clavulanic Acid (generic)

Semi-synthetic Amoxicillin – bacterial cell wall synthesis

Clavulanic Acid – inactivates some beta-lactamase enzymes

Sinus, Ear, Respiratory, Skin, and Urinary Infections
Cipro (brand name)  Ciprofloxacin (generic) Synthetic Nucleic acid synthesis – bacterial DNA gyrase Urinary, Skin, Bone, Joint, Sexually Transmitted, Respiratory, Gastrointestinal Infections
Bactrim (brand name)  Sulfamethoxazole and Trimethoprim (generic) Antibiotic Metabolic pathways – folic acid synthesis Minor cuts, scrapes, and burns, Meningitis, Respiratory, Urinary Infections, and Sepsis
Neosporin (brand name, topical)

Poly-Rx (brand name, systemic)

Polymyxin B (generic, systemic)

Synthetic Disrupts membranes Urinary, Ear, Respiratory, and Gastrointestinal Infections



1. Measure the diameter of the zone of inhibition from one edge to the other; right over the middle of the antibiotic disk. If the bacteria grow right up to the disk, the zone of inhibition is reported as “0 mm.” This indicates that the bacteria are resistant to that antibiotic. If mutant strains are growing inside the general zone of inhibition or if the edges of the zones are “fuzzy”, measure the innermost clear zone. Record the results on the worksheet.

Measuring the zones of inhibition

Kirby Bauer Sensitivity Test
Kirby Bauer Test zone of inhibition is 32mm

2.  The zone of inhibition is the result of two factors: how quickly the organism grows and how fast the antibiotic diffuses through the media. Since antibiotics have different molecular weight and therefore different diffusion rates, antibiotics cannot be compared to each other. Instead, each antibiotic zone size must be compared to a standardized chart. Compare the zone sizes of your zones to the standardized chart on the worksheet. Determine If the bacteria is sensitive, intermediate, or resistant to each antibiotic. Record your results on the worksheet.

3. Using the S, I, R results for the tested bacteria, complete the spectrum of activity chart in the worksheet.


1. Determine the MIC for the E-Test plates results shown on the worksheet. The MIC value is read from the scale in terms of µg/mL where the ellipse edge intersects the strip. Examples of E- Test results and interpretation are shown below.

Examples of MIC results

2. Record the MIC results in the worksheet.

Demonstration of Beta-Lactamase Activity

You will determine if one of the organisms used in this lab exercise can synthesize the beta-lactamase enzyme. Beta-lactamase destroys the beta-lactam ring of penicillin. The end product will be penicilloic acid.

Beta-lactam ring of penicillin
The chemical structure of penicillin. Beta-lactamase enzymes target and eventually break down the beta-lactam ring (shown in red).

Beta-Lactamase tube

1.  The instructor added approximately 5 drops of deionized water to the tube containing penicillin and a pH indicator. Using a sterile, cool inoculating wire, a heavy inoculum was used to make a suspension of organisms in the tube.

2.  Observe the tube for a color change (pink to yellow).

Yellow = Positive (due to the acidic pH change from production of penicilloic acid)

Beta-Lactamase Test Negative Result
Beta-Lactamase Test Negative Result

Pink = Negative (no color change because no penicilloic acid was produced)

Beta-Lactamase Test Positive Result
Beta-Lactamase Test Positive Result

3.  Answer a few questions about the beta-lactamase demonstration on the worksheet.


Photo of Dr. Jane Hinton


American veterinarian Dr. Jane Hinton helped create Muller Hinton agar in 1941. It is non-selective, non-differential medium, so almost all bacteria can grow on it. The starch in the media absorbs toxins released from the bacteria, so they do not interfere with the antibiotic. It is a loose agar; this allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a more accurate zone of inhibition.


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Laboratory Exercises in Microbiology Copyright © 2022 by Anne Mason M.S. and Jill Raymond Ph.D. is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.

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