UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE

Kirby Bauer Antibiotic

Sensitivity Test
Pharmaceutical Microbiology Assignment

Qasim Abbas
BS (Hons.) Applied Microbiology
Semester: 7th
Reg. No: 2013-am-029
Department of Microbiology

Kirby Bauer Antibiotic Sensitivity Test

TABLE OF CONTENTS
INTRODUCTION 2
PRINCIPLE 3
MATERIALS REQUIRED .. 3
PROCEDDURE 4
PLATE OBESRVATION ..... 5
MEASUREMENT OF ZONE OF INHIBITION . 5
RESULTS TABLE .. 6

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Kirby Bauer Antibiotic Sensitivity Test

INTRODUCTION
Certain bacteria can display resistance to one or more antibiotics. Determining
bacterial antibiotic resistance whether a bacterium can survive in the presence of
an antibiotic - is a critically important part of the management of infectious diseases
in patients. The Kirby-Bauer (K-B) disk diffusion test is the most common method
for antibiotic resistance/susceptibility testing. The results of such testing help
physicians in choosing which antibiotics to use when treating a sick patient.
The Kirby-Bauer (K-B) test utilizes small filter disks impregnated with a known
concentration of antibiotic. The disks are placed on a Mueller-Hinton agar plate that
is inoculated with the test microorganism. Upon incubation, antibiotic diffuses from
the disk into the surrounding agar. If susceptible to the antibiotic, the test organism
will be unable to grow in the area immediately surrounding the disk, displaying a
zone of inhibition (see figure below). The size of this zone is dependent on a number
of factors, including the sensitivity of the microbe to the antibiotic, the rate of
diffusion of the antibiotic through the agar, and the depth of the agar.
Microorganisms that are resistant to an antibiotic will not show a zone of inhibition
or display a relatively small zone.

Zone of Inhibition

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Kirby Bauer Antibiotic Sensitivity Test

PRINCIPLE
Antibiotic discs are placed over the lawn of test organism on the surface of media. So
during incubation the antibiotic agent diffuses down into the media and will interfere
with the growth on inoculated test organism. If the antibiotic is effective against that
particular organism it will inhibit the growth of organism and a zone of inhibition
will be produced around antibiotic disc. Zone diameter is measured and the
effectiveness of that antibiotic against a particular organism is determined by this
sensitivity assay.

MATERIALS REQUIRED
Media
Plates of Muller Hinton agar

Test Organism
Stock broth culture of test organism should be available such as Escherichia coli
(gram -ve) or Staphylococcus epidermidis (gram +ve) .

Antimicrobial sensitivity discs

Different antibiotic discs like penicillin, gentamicin and tetracyclin of different
concentration according to requirements.

Equipment
1) Disc dispenser or forceps
2) Bunsen burner
3) Sterile cotton swabs
4) Glassware marking pencil
5) Millimeter ruler
6) Test tube rack

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Kirby Bauer Antibiotic Sensitivity Test

PROCEDURE
1) Label the agar plates with the culture organism being used also marks dots at
comparatively same distance where you will place the antibiotic discs.
a. Discs should be minimum 20 mm apart from each other
b. Discs should not be placed near the edge of the plate
2) Inoculate the plate with your test bacterium as follows
a. Using aseptic technique, wet a swab with the broth culture
b. Thoroughly swab the surface of the plate, making sure to cover the
entire surface (1st swabbing)
c. Do not get more culture
d. Turn the plate approximately 60 degree and repeat the previous step
(2nd swabbing)
e. Do not get more culture
f. Repeat the previous step (3rd swabbing)
g. Use the following pattern for swabbing

3) Discard the swab in bleach containing beaker

4) Place one antibiotic disc on the surface of the agar, using aseptic technique as
follows
a. Heat the tips of the forceps by placing them just inside the opening of
bacterial incinerator for 5-10 seconds
b. Carefully pick up your test disc with forceps and gently place it on the
appropriate spot on the agar plate
c. To ensure that the disc is flat on the agar, gently press it down with
forceps
d. Reheat the tips of forceps as above to kill any bacteria
5) Repeat the procedure for the second antibiotic procedure
6) Repeat the steps 1-6 on second agar plate with the new bacterium
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Kirby Bauer Antibiotic Sensitivity Test

7) Wait until the surface of the plates has completely dried
8) Incubate both plates at 37C for 24 hours

PLATES OBSERVATION
Observe the agar plates after incubation. For positive antibiotic disc there will be
inhibited microbial growth around that disc. Zone of inhibition will be produced.
Observe it carefully and measure the diameter of the zone to evaluate the
effectiveness of that antibiotic against particular organism.
Antibiotic will diffuse in the media and will cause the inhibition of bacterial growth if
effective against test organism, otherwise there will be no inhibition of growth and
test organism will show the adequate growth around antibiotic disc as well.

MEASUREMENT OF ZONE OF INHIBITION

If zone is present, measure the diameter of zone in mm and record your
results in the table.
If there is no zone, record your result as 0 mm.
Compare the results
See following images

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Kirby Bauer Antibiotic Sensitivity Test

RESULTS TABLE
Sizes of zone of inhibition (in mm)
Test
Organism
inoculated

Antibiotics & their concentrations

Diameter Measured (mm)

Penicillin G Tetracyclin

gentamicin

Penicillin
G

Tetracyclin

Gentamicin

Escherichia coli

10 ug

30 ug

10 ug

Staphylococcus
epidermidis

10 ug

30 ug

10 ug

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