The Evaluation of Antibiotics Using Kirby
Bauer Disk Diffusion Method
ACKNOWLEDGMENTS: Source document adapted from: A Laboratory Manual for Microbiology, Third
ed., by John M. Larkin, with some additions by Ruth A. Gyure for WISTR workshop July 2006, Western
CT State University
URL source: http://www.waksmanfoundation.org/labs/lsu/antibio.html
Note: The lab is part of the Waksman Foundation collection of microbiology resources for K-12 teachers,
an excellent resource, starting page found at:
http://www.waksmanfoundation.org/html/resource_k12.html
Photo of plate above courtesy of University of New South Whales Medical School website.
INTRODUCTION: Antibiotics are chemicals that are produced by living organisms which, even in minute
amounts, inhibit the growth of or kill another organism. While thousands of them have been discovered
since Sir Alexander Fleming observed the inhibitory activity of Penicillium on Staphylococcus in 1929,
most are not useful medically because of undesirable toxicity or side effects. A simple method using
paper discs saturated with the chemicals to be tested can be used to determine the resistance or
sensitivity of an organism to different antibiotics. Following inoculation of an agar medium, discs are
placed onto the surface of the medium so that the antibiotic will diffuse into the medium. Inhibition of the
organism is evident following incubation as a clear region around the disc, called a "zone of inhibition", in
which no growth has occurred.
The size of the zone of inhibition is determined by the type of medium used, the solubility and rate of
diffusion of the antibiotic, the amount of inoculum, as well as the effect of the antibiotic. This experiment
simulates a commonly used clinical technique called the Kirby-Bauer method in which exact
specifications must be followed. Numerous antibiotics are tested for usefulness against the actual
organism causing a patient's illness. Medical laboratory personnel select the antibiotic discs tested based
upon the site and type of infection. Following incubation and measurement of the zone sizes, a chart is
consulted which indicates whether the diameter of the zone of inhibition for an antibiotic indicates that it
would be effective for use in treating the patient's infection.
Materials per group of 4 students:



plates of Trypticase Soy Agar (TSA) or Nutrient Agar (NA)
sterile swabs
Overnight broth cultures of Staphylococcus aureus (Sa), and Pseudomonas aeruginosa (Pa) or
BSL level 1 organisms such as Micrococcus luteus, Escherichia coli, Staphylococcus epidermidis,
Materials to be shared by the class:



Dispensers of antibiotic discs
Beakers of disinfectant
Sterile toothpicks or applicator sticks
Protocol:
1. Dip one swab into the broth of Pseudomonas.
2. Swab the entire surface of one plate labeled, "Pa". Go over the plate at least twice in each
direction. Discard the swab in the beaker of disinfectant provided.
3. Repeat this procedure for the Staphylococcus.
4. Add the antibiotic discs to each plate using the dispenser. Remove the cover of the petri dish and
set the dispenser over the plate. If the dispenser is not dispensing discs, consult the instructor.
5. Gently tap each antibiotic disc onto the surface of the agar with a sterile stick or toothpick to
assure good contact. Discard toothpicks in the disinfectant.
6. Incubate plates at 37EC until the next lab period.
7. Examine each plate for the presence of zones of inhibition.
8. Measure the diameter of each zone in millimeters using the rulers provided.
9. Compare the diameter of each zone to the chart to determine if the organism is sensitive or
resistant to the antibiotic.
Results of Antibiotic Sensitivities:
Organism______________
Antibiotic
Abbreviation
on disc
Streptomycin
Ampicillin
Gentamicin
Erythromycin
Chloramphenicol
Tetracycline
S10
AM
GM
E
C
Te
Zone
Diameter
Resistant
Diameter
Intermed
Diameter
Sensitive
Diameter
11 mm
11 mm
12 mm
13 mm
12 mm
14 mm
12-14 mm
12-14 mm
13-14 mm
14-22 mm
13-17 mm
15-18 mm
15 mm
15 mm
15 mm
23 mm
18 mm
19 mm
Sensitive
Resistant
Results of Antibiotic Sensitivities
Organism______________
Antibiotic
Abbreviation
on disc
Streptomycin
Ampicillin
Gentamicin
Erythromycin
Chloramphenicol
Tetracycline
S10
AM
GM
E
C
Te
Zone
Diameter
Resistant
Diameter
Intermed
Diameter
Sensitive
Diameter
11 mm
11 mm
12 mm
13 mm
12 mm
14 mm
12-14 mm
12-14 mm
13-14 mm
14-22 mm
13-17 mm
15-18 mm
15 mm
15 mm
15 mm
23 mm
18 mm
19 mm
Sensitive
Resistant
Teacher's instructions for The Evaluation of
Antibiotics
The teacher must inoculate tubes of Trypticase Soy broth (or nutrient broth) with Staphylococcus aureus
and Pseudomonas aeruginosa or other culture 24-48 hours before the class. Remember to practice your
best aseptic technique while inoculating the cultures. The cultures will be incubated at about 37*C until
needed.
While the cultures can be used for more than one class, the potential for contamination of the culture will
increase with use. For this reason, you are provided four tubes of broth per class. Per class, inoculate two
tubes with Staphylococcus aureus and two tubes with Pseudomonas aeruginosa or other organism used.
Procedure for making the starting cultures:
1. Start with sterile tubes of Trypticase Soy broth or Nutrient Broth. It is safest to AUTOCLAVE
media but media boiled for 10 minutes or so will work for the procedure. Be sure to transfer media
carefully and sterilely into the tubes, which would be best if disposable plastic (purchased sterile).
Label the tubes with names of organisms. Again, we suggest inoculating a separate starting tube
for each group of students and for each class.
2. Organize your stock plates of organisms (stored at RT or in refrigerator with parafilm seals or in a
sealed box with humidity source).
3. Choose one of the plates. Use a sterile disposable inoculation loop to place a visible amount of
the bacteria from the surface of the plate into the correctly labeled tube of broth. Do not set the
loop down. You will use the same loop to inoculate other tubes of broth with the same bacterium.
Discard of the loop in the biohazard bag or your beaker of bleach or disinfectant. Cultures can be
inoculated with sterile sticks but a disposable loop is simpler.
4. Snap or screw all the tube caps down tight. Holding the tube just below the cap, thump the
bottom of the tube with your other hand to suspend the bacteria in the broth. Loosen caps slightly.
5. Incubate the tubes upright in the rack at approximately 37*C until class. Usually 24-48 hours is
adequate for abundant growth. The broth in the tubes should be cloudy if the bacteria grew.
Some excellent supplementary PHOTOS of results of disk diffusion assay, from Albert Einstein College of
Medicine: http://gold.aecom.yu.edu/id/micro/kirbybauer.htm
ENRICHMENT:
1. An added activity from ASM Microbelibrary is provided, allowing students to use statistical methods to
compare replicate samples from multiple groups:
Using a Disk Diffusion Assay to Introduce Statistical Methods
2. Also see 'Cultured Inquiry,' an article from NSTA journal. This describes how to develop a true inquiry
based laboratory allowing students to test their own compounds for antibiotic effects on microbes.