ANTIMICROBIAL SUSCEPTIBILITY TEST

(Kirby – Bauer Method)

The number of antibiotics and other antimicrobials available today is larger

than ever before. New antibiotics are continuously being developed and discovered;
thus there is an increasing demand on the clinical laboratory to determine the
antibiotic susceptibility or resistance of various pathogenic bacteria.

One method that will be illustrated in this experiment is the sensitivity disk
method of Kirby-Bauer Method. In this method, antibiotics are impregnated onto
paper disks and then placed on a seeded Mueller Hinton Agar Plate using a
mechanical dispenser or sterile forceps. The plate is then incubated for 16-18 hours,
and the diameter of the zone of inhibition around the disk is measured to the nearest
millimeter. The inhibition zone diameter that is produced will indicate the
susceptibility or resistance of a bacterium to the antibiotic. This can be determined by
comparing the zone diameter with the known zone diameter size for susceptibility. For
example, a zone of a smaller diameter or no zone at all show that the bacterium is
resistant to the antibiotic.

Many factors are involved in sensitivity disk testing and must be carefully
controlled. These include: size of the inoculums, distribution of the inoculums,
incubation period, depth of the agar, diffusion rate of the antibiotic, concentration of
antibiotic in the disk, and growth rate of the bacterium. If all of these factors are
carefully controlled, this type of testing is highly satisfactory for determining the
degree of susceptibility of a bacterium to a certain antibiotic.

The Kirby-Bauer Method is not restricted to antibiotics. It may also be used to

measure the sensitivity of any microorganism to a variety of antimicrobial agents such
as sulfonamides and synthetic chemotherapeutics.

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 A Kirby-Bauer Plate

Notice the diameter of the various zones of inhibition

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Interpretation of the Zones of Inhibition of Antibiotics on Test Cultures

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 Acceptable Limits for Quality Control Strains Used to Monitor
Accuracy of Disk Diffusion Testing of Non fastidious Organisms
(Using Mueller-Hinton Medium Without Blood or Other Supplements)

Antimicrobi Escherichia Staphylococcus Pseudomonas Escherichia

Disk coli aureus aeruginosa coli
al
Content ® b ® ® ® f
Agent ATCC 25922 ATCC 25923 ATCC 27853 ATCC 35218
Amikacin 30 μg 19–26 20–26 18–26 –
Amoxicillin-
clavulanic 20/10 μg 18−24 28–36 – 17−22
acid
Ampicillin 10 μg 16−22 27–35 – 6
Ampicillin-
10/10 μg 19–24 29–37 – 13–19
sulbactam
Azithromycin 15 μg – 21–26 – –
Azlocillin 75 μg – – 24–30 –
Aztreonam 30 μg 28–36 – 23–29 –
Carbenicillin 100 μg 23–29 – 18–24 –
Cefaclor 30 μg 23–27 27–31 – –
Cefamandole 30 μg 26–32 26–34 – –
Cefazolin 30 μg 21–27 29–35 – –
Cefdinir 5 μg 24–28 25–32 – –
Cefditoren 5 μg 22–28 20–28 – –
Cefepime 30 μg 31−37 23–29 24–30 –
Cefetamet 10 μg 24–29 – – –
Cefixime 5 μg 23–27 – – –
Cefmetazole 30 μg 26–32 25–34 – –
Cefonicid 30 μg 25–29 22–28 – –
Cefoperazone 75 μg 28–34 24–33 23–29 –
Cefotaxime 30 μg 29–35 25–31 18–22 –
Cefotetan 30 μg 28–34 17–23 – –
Cefoxitin 30 μg 23–29 23–29 – –
Cefpodoxime 10 μg 23–28 19–25 – –
Cefprozil 30 μg 21–27 27–33 – –
Ceftazidime 30 μg 25–32 16–20 22-29 –
Ceftibuten 30 μg 27–35 – – –
Ceftizoxime 30 μg 30–36 27–35 12–17 –
Ceftriaxone 30 μg 29–35 22–28 17–23 –

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 (Cont.)
Acceptable Limits for Quality Control Strains Used to Monitor
Accuracy of Disk Diffusion Testing of Non fastidious Organisms
(Using Mueller-Hinton Medium Without Blood or Other Supplements)

Antimicrobi Escherichia Staphylococcus Pseudomonas Escherichia

Disk coli aureus aeruginosa coli
al
Content ® b ® ® ® f
Agent ATCC 25922 ATCC 25923 ATCC 27853 ATCC 35218

Cefuroxime 30 μg 20–26 27–35 – –

Cephalothin 30 μg 15–21 29–37 – –
Chlorampheni
30 μg 21–27 19–26 – –
col
Cinoxacin 100 μg 26–32 – – –
Ciprofloxacin 5 μg 30–40 22–30 25–33 –
Clarithromyci
15 μg – 26–32 – –
n
Clinafloxacin 5 μg 31–40 28–37 27–35 –
Clindamycin 2 μg – 24–30 – –
d
Daptomycin 30 μg – 18–23 – –
Dirithromycin 15 μg – 18–26 – –
Doxycycline 30 μg 18–24 23–29 – –
Enoxacin 10 μg 28–36 22–28 22–28 –
Ertapenem 10 μg 29–36 24–31 13–21 –
Erythromycin 15 μg – 22–30 – –
Fleroxacin 5 μg 28–34 21–27 12–20 –
c
Fosfomycin 200 μg 22–30 25–33 – –
Garenoxacin 5 μg 28-35 30-36 19-25 –
Gatifloxacin 5 μg 30–37 27–33 20–28 –
Gemifloxacin 5 μg 29–36 27–33 19–25 –
a
Gentamicin 10 μg 19–26 19–27 16–21 –
Grepafloxacin 5 μg 28–36 26–31 20–27 –
Imipenem 10 μg 26–32 – 20–28 –
Kanamycin 30 μg 17–25 19–26 – –
Levofloxacin 5 μg 29–37 25–30 19–26 –
Linezolid 30 μg – 25–32 – –
Lomefloxacin 10 μg 27–33 23–29 22–28 –
Loracarbef 30 μg 23–29 23–31 – –
Mecillinam 10 μg 24–30 – – –

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 Laboratory Exercise
ANTIMICROBIAL SUSCEPTIBILITY TEST
(Kirby – Bauer Method)

Objectives:

At the end of the activity, the students will be able to:

1. appreciate the scope of antimicrobial activity of selected antibiotics;

2. perform the Kirby-Bauer Method for determining antibiotic sensitivity; and
3. correctly interpret a Kirby-Bauer plate.

Materials:

Mueller Hinton (MH) plated medium

100 mm Petri dish
Sterile cotton swab
Incubator
Forceps
Vernier caliper/ruler
Bunsen burner / alcohol lamp
Antibiotic disks
McFarland standard

Procedure:

A. Plate Preparation
1. Agar depth: 4-6 mm
2. For 150 mm Petri dish: 70-80 mL medium
3. For 100 mm Petri dish: 20-25 mL medium
4. Prepared plates should be stored at 4 0C in cellophane wrapping and
should be used within a 2-week period.
5. Immediately prior to use, plates should be “dried” in an incubator for 30
minutes to facilitate removal of excess surface moisture.

B. Inoculum Preparation
1. Using an inoculating needle touch 4 or 5 colonies of the organism and
inoculate into 4-5 ml of suitable broth medium (TSB).
2. Incubate the broth at 35 0C until the turbidity of the culture compares to
that of the recommended turbidity standard (McFarland standard)
prepared by adding 0.5 ml of 0.048 M BaCl2 and 99.5 ml 0.36 N H2SO4.
The standard should be agitated on a vortex mixer immediately prior to

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 use. Unless a standard is contained in heat-sealed glass tubes it should
be replaced every 6 months. If appropriately sealed, the standard may
last indefinitely.
3. Using a white paper with horizontal black lines, compare the turbidity. If
it is more turbid than the standard, it may be adjusted by adding sterile
saline or broth; if it is less turbid, add colonies, provided they are well
isolated and are morphologically indistinguishable from those originally
selected.
4. The standard inoculums suspension should be inoculated within 20-25
minutes.

C. Medium Inoculation and Disk Placement

1. Using a sterile cotton swab, immerse it into the standardized inoculums
suspension.
2. Express the excess broth by pressing and rotating the swab against the
inside of the suspension tube.
3. Streak the swab evenly in 3 directions on the surface of the agar plate.
4. Make a final circular motion around the agar rim with the cotton swab.
5. Dry the inoculums for 3-5 minutes and then apply the disks, either by a
mechanical dispenser or by hand using sterile forceps.
6. After placing the disks, press firmly and gently to the agar surface.
7. The spatial arrangement of the disks should be such as to obviate the
development of overlapping zones of inhibition.
8. Incubate overnight.

D. Interpretation
1. Measure the zone diameters with a metric ruler on the undersurface of
the Petri dish under reflected light.
2. If blood has been added to the Mueller Hinton agar, the zones are
measured from the surface of the agar after removing the cover.
3. The endpoint is complete inhibition of growth as determined visually,
except in the case of sulfonamides where organisms may grow through
several generations before inhibitions occur.

RESULT:

Disk Symbol Antibiotic Name Diameter (mm) of Interpretation

Zone of Inhibition (S, I, R)

1. _________ ______________ ___________ ___________

2. _________ ______________ ___________ ___________
3. _________ ______________ ___________ ___________
4. _________ ______________ ___________ ___________
5. _________ ______________ ___________ ___________

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 Laboratory Exercise

ANTIMICROBIAL SUSCEPTIBILITY TEST

(Kirby – Bauer Method)

Name: _____________________________________________ Date Performed: ____________

Course, Yr. & Sec: _________________________________ Date Submitted:_____________
Group No:________________ Instructor: _____________________

Instruction:

Assuming that below is the result form of your laboratory, fill in the needed
information based on the results of your sensitivity testing.

Name ___________________________________ Age: ___________Sex: _____________________

Doctor: __________________________________ Date: ___________Time Specimen Taken: _______

Specimen: _________________________________________________ Organism: _____________________

Date Reported: ______________________________________________ Colony Count: __________________

ANTIBIOTIC SENSITIVITY TEST

DISK DISK INTERPRETATION

SYMBOL NAME Sensitive Intermediate Resistant
(s) (I) (R)

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Guide Questions:

1. Why would a Clinical Microbiology laboratory perform antimicrobial

susceptibility tests? (2pts)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

2. How do you think the result could help physicians in the diagnosis of
their patient? (2pts)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

3. What are the problems encountered in the interpretation of Kirby-

Bauer Technique and how would you remedy each problem? (2pts)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

4. How do you interpret result in Kirby-Bauer Antimicrobial Susceptibility

Test? (2pts)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

5. How would you standardize the inoculums in the above technique?

(2pts)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

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