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Laboratory Exercise 16

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 array of
antibiotics.
Materials:
Mueller-Hinton agar plates; Cultures of: Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Enterococcus faecalis and Mycobacterium phlei; Antibiotic
disks: Ampicillin, Ceftriaxone, Cefurixome, Ciprofloxacin, Doxycyline, Erythromycin,
Gentamicin, Oxacillin, Penicillin G, Sulfamethoxazole; 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 35C 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 ff. criteria:

Diameter of Zone of
Inhibition (mm)
Resistan Intermedi Sensiti
t
ate
ve

Antibiotic

Symb
ol

Disc
Concentra
tion

Streptomycin

10 g

11

12-14

15

Tetracycline

Te

30 g

14

15-18

19

Nalidixic Acid

NA

30 g

13

14-18

19

Chloramphenicol

30 g

12

13-17

18

Rifampin

RA

5 g

16

17-19

20

Ampicillin

AM

10 g

13

14-16

17

Ceftriaxone

CRO

30 g

19

20-22

23

Cefuroxime

CXM

30 g

15

15-17

18

Ciprofloxacin

CIP

5 g

15

16-20

21

Doxycycline

DO

30 g

12

13-15

16

Erythromycin

15 g

<13

14-22

>23

Gentamicin

CM

10 g

12

13-14

15

Oxacillin

OX

1 g

<10

11-12

>13

Penicillin

10 g

Staphylococcus

28

29

Enterococcus

14

15

Streptococcus

19

20-27

28

Others

19

20

10

11-15

16

Trimethoprin/Sulfamethox
azole

SXT

25 g

NAME
S:

DATE
PERFORMED:

DATE
SUBMITTED:

Laboratory Exercise No. 16


ANTIMICROBIAL SUSCEPTIBILITY TESTING
(Kirby-Bauer Procedure)
RESULTS:
(Directly fill up the tables. Be sure to indicate what test microorganism was

used!)
* Pictures/Figures should be presented as appendix/ces.
Sample Table:
Gram Negative
Chemotherape
utic Agent

Escherichia coli
Average
zone size
(mm)

Pseudomonas aeruginosa

Susceptibilit
y

Average
zone size
(mm)

Susceptibilit
y

Ampicillin (AM)
Ceftriaxone
(CRO)
Cefuroxime
(CXM)
Ciprofloxacin
(CIP)
Doxycycline
(DO)
Erythromycin
(E)
Gentamicin (CN)
Oxacillin (OX)
Penicillin G (P)
Trimethoprin/
Sulfamethoxazo
le (SXT)

Gram Positive
Chemotherape
utic Agent

Ampicillin (AM)

Staphylococcus aureus
Average
zone size
(mm)

Susceptibilit
y

Bacillus subtilis
Average
zone size
(mm)

Susceptibilit
y

Ceftriaxone
(CRO)
Cefuroxime
(CXM)
Ciprofloxacin
(CIP)
Doxycycline
(DO)
Erythromycin
(E)
Gentamicin (CN)
Oxacillin (OX)
Penicillin G (P)
Trimethoprin/
Sulfamethoxazo
le (SXT)

Chemotherapeuti
c Agent

Acid-fast: Mycobacterium phlei


Average zone size (mm)

Susceptibility

Ampicillin (AM)
Ceftriaxone (CRO)
Cefuroxime (CXM)
Ciprofloxacin (CIP)
Doxycycline (DO)
Erythromycin (E)
Gentamicin (CN)
Oxacillin (OX)
Penicillin G (P)
Trimethoprin/
Sulfamethoxazole
(SXT)
DISCUSSION
State your interpretation of results. End the discussion by summarizing major
conclusions.
Study Questions:
Short but concise answers must be placed after every question.
Cite your references.
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 McFarlands 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?