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Laboratory Exercise 8
ANTIMICROBIAL SUSCEPTIBILITY TESTING
(Kirby-Bauer Procedure)

The purpose of the Kirby-Bauer disk diffusion susceptibility test is to determine the
sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various
antimicrobial compounds in order to assist a physician in selecting treatment options for his or
her patients. The pathogenic organism is grown on Mueller-Hinton agar in the presence of
various antimicrobial impregnated filter paper disks. The presence or absence of growth around
the disks is an indirect measure of the ability of that compound to inhibit that organism.

Objectives: At the end of the experiment, the student must be able to:
1. Perform the agar-disk diffusion method of antimicrobial susceptibility testing;
2. Determine the sensitivity of different species of bacteria to (an) antibiotic/s.

Materials:
Mueller-Hinton agar plates; 0.5 McFarland Turbidity Standard, Tubes with 0.85% Saline Solution;
Cultures of: Escherichia coli, Staphylococcus aureus Antibiotic disks, alcohol lamps, sterile
cotton swab, forceps, caliper or ruler

Procedure:
1. Using sterile techniques, inoculate the organisms into the Mueller-Hinton agar plate as
follows:
a. Gently agitate your broth tube to resuspend any bacteria that have settled to the bottom
of the tube.
b. Dip a sterile cotton swab into a well-mixed saline solution of test culture.
c. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to
remove excess fluid. The swab should not be dripping wet.
d. Completely swab the surface of the M-H plate. Rotate the plate 90° and repeat, dipping a
fresh sterile swab into the broth tube and completely swabbing the surface of the plate.
Rim the plate with the swab by running the swab around the edge of the entire the plate
to pick up any excessive inoculum that may have been splashed near the edge. There
should be a blanket of uniform growth following incubation.
2. Allow all the plates to dry for about 5 minutes.
3. Using a pair of forceps, obtain a disk from the antibiotic cartridge and place it on the surface
of the agar. Place the disks at equal distances from each other. Gently press the disks with
the forceps or with an applicator stick to ensure adherence of the disk into the agar. Always
sterilize the forceps before obtaining an antibiotic disk and after placing the disk in the agar
plate, by dipping it in an alcohol and flaming it over the alcohol lamp.
4. Incubate the plates at 35°C for 24 hours.
5. Using a ruler or calliper and while, measure the diameter of the zone of inhibition to the
nearest millimeter after incubation.
 When measuring zone diameters, always round up to the next millimeter.
 All measurements are made with the unaided eye while viewing the back of the petri
dish. Hold the plate a few inches above a black, non-reflecting surface illuminated with
reflected light.
6. Determine the susceptibility of the organisms using the following criteria:
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Diameter of Zone of Inhibition

Disc
Symbo (mm)
Antibiotic Concentratio
l Resistan Intermediat Sensitiv
n
t e e
Streptomycin S 10 µg ≤ 11 12-14 ≥ 15

Tetracycline Te 30 µg ≤ 14 15-18 ≥ 19

Nalidixic Acid NA 30 µg ≤ 13 14-18 ≥ 19

Chloramphenicol C 30 µg ≤ 12 13-17 ≥ 18

Rifampin RA 5 µg ≤ 16 17-19 ≥ 20

Ampicillin AM 10 µg ≤ 13 14-16 ≥ 17

Ceftriaxone CRO 30 µg ≤ 19 20-22 ≥ 23

Cefuroxime CXM 30 µg ≤ 15 15-17 ≥ 18

Ciprofloxacin CIP 5 µg ≤ 15 16-20 ≥ 21

Doxycycline DO 30 µg ≤ 12 13-15 ≥ 16

Erythromycin E 15 µg <13 14-22 >23

Gentamicin CM 10 µg ≤ 12 13-14 ≥ 15

Oxacillin OX 1 µg <10 11-12 >13

Penicillin P 10 µg

Staphylococcus ≤ 28 - ≥ 29

Enterococcus ≤ 14 - ≥ 15

Streptococcus ≤ 19 20-27 ≥ 28

Others ≤ 19 - ≥ 20
Trimethoprin/Sulfamethoxazol
SXT 25 µg ≤ 10 11-15 ≥ 16
e
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RESULTS and DISCUSSION

Directly fill up the tables. Be sure to indicate what test microorganism was used!
State your interpretation of results. End the discussion by summarizing major conclusions.

* Pictures/Figures should be presented as appendix/ces.

Sample Table:
Bacteria

Chemotherapeutic Escherichia coli S. aureus

Agent Average
Average zone
zone size Susceptibility Susceptibility
size (mm)
(mm)

Tetracycline

STUDY QUESTIONS:
1. What factors affect the zone of inhibition?
2. What is the action of each chemotherapeutic agent? Which chemotherapeutic agent is the
most effective? The least effective? (to each test organism)
3. In which growth phase is the organism most sensitive to the antibiotic?
4. What are other methods of measuring the sensitivity of organisms to antibiotic?
5. What is MIC and MBC? How are they measured?
6. What is being measured in the agar-disk diffusion method, bactericidal or bacteriostatic
activity of the organism?
7. Why is the agar-disk diffusion method not a perfect indication on how well the
chemotherapeutic agent will perform in vivo?
8. What is McFarland’s Standards? How is it useful to antibiotic sensitivity test?
9. Why is Mueller Hinton Agar used in this test and not other ordinary agar media?