ANTIMICROBIAL SUSCEPTIBILITY TESTING USING KIRBY-BAUER METHOD

Objective

 To be able to perform a virtual laboratory on antimicrobial susceptibility testing using

Kirby-Bauer method.
 To be able to determine the antimicrobial susceptibility of a given bacterial isolate.
 To be able to determine the importance of Kirby-Bauer test in clinical laboratory.
 To be able to determine how the Kirby Bauer test is standardized.
 To be able to describe the Kirby- Bauer disk diffusion susceptibility test protocol

Theory

Determination of bacterial resistance to antimicrobials is an important part of the

management of infections in patients.   The Kirby-Bauer test, known as the disk-diffusion
method, is the most commonly used method for initial susceptibility testing in clinical samples.
This method relies on the inhibition of bacterial growth measured under standard conditions. For
this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly and aseptically
inoculated with the test organism and then filter paper discs, which are impregnated with a
specific concentration of a particular antibiotic, are placed on the medium. After allowing the
bacteria to grow overnight, areas of clear media surrounding the disks indicate that the antibiotic
inhibits bacterial growth. The concentration of antibiotic that diffuses into the media decreases
with increasing distance from the source. Therefore, the more sensitive the bacteria are to a
given antibiotic, the larger the clear bacteria-free zone that forms around the disk containing that
antibiotic.

In this method, the bacterium is swabbed on the agar and the antibiotic discs are placed
on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is
away from the disc.  If the organism is susceptible to a specific antibiotic, there will be no growth
around the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and
measured to determine the susceptibility to an antibiotic for that particular organism. The
measurement is compared to the criteria set by the Clinical and Laboratory Standards Institute
(CLSI). Based on the criteria, the organism can be classified as being Resistant (R),
Intermediate (I) or Susceptible (S).

Materials

 Petri dish
 Mueller-Hinton agar plate
 Bacterial isolate (E. coli)
  Antibiotic discs
 Cotton swabs
 Petri dishes
 0.5 McFarland Turbidity standard
 Inoculum
 Forceps
 Incubator
 Metric ruler
 Kirby Bauer Table of interpretative standard

Procedures

1. Prepare the inoculum from the primary culture plate by touching with a loop the
tops of each of 3 – 5 colonies, of similar appearance, of the organism to be
tested and transfer this growth to a tube of saline. If the inoculum has to be made
from a pure culture,  suspend a loopful of the confluent growth similarly.
2. Compare the tube with the 0.5 McFarland turbidity standard (approx cell density
1.5 x10^8 CFU/ml) and adjust the density of the test suspension to that of the
standard by adding more bacteria or more sterile saline.
Remember: Proper adjustment of the turbidity of the inoculum is essential to
ensure that the resulting lawn of growth is confluent or almost confluent.
3. Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess
inoculum by pressing and rotating the swab firmly against the side of the tube
above the level of the liquid.
4. Streak the swab all over the surface of the medium three times, rotating the plate
through an angle of 60 ° after each application. Finally, pass the swab around the
edge of the agar surface. The swab should follow as it is drawn across the plate
(as shown in the figure). #Discard the swab into an appropriate container.
5. Leave the inoculum to dry for a few minutes (at least 3 to 5 minutes, but no more
than 15 minutes) at room temperature with the lid closed.
6. Place the appropriate antimicrobial-impregnated disks on the surface of the agar
(antimicrobial disks can be purchased from any reputable suppliers)
7. Antimicrobial discs can be placed on the inoculated plates using a pair of sterile
forceps. It is convenient to use a template to place the discs uniformly or a sterile
needle-tip may also be used to place the antibiotic discs on the plate.
Alternatively, an antibiotic disc dispenser (as shown in image-2) can be used to
apply the discs to the inoculated plate.
8. Disks should not be placed closer than 24 mm (center to center) on the Muller
Hinton agar plate.  Ordinarily, no more than 12 disks should be placed on a 150-
mm plate or more than 5 disks on a 100-mm plate. #avoid placing disks close to
the edge of the plate as the zones will not be fully round and can be difficult to
measure.
9. Each disc should be gently pressed down to ensure complete contact with the
agar surface and do not fall when the plate is inverted during incubation. #Do not
push the disc into the agar.
10. The plates should be placed in an incubator at 35 °C within 30 minutes of
preparation. Temperatures above 35 °C invalidate results for oxacillin/methicillin.
 #Do not incubate in an atmosphere of carbon dioxide, this will decrease the pH of
the agar and result in errors due to incorrect pH of the media.
11. Examine the plate after 16-24 hours incubation.

Reading and Interpreting Zone Sizes

1. Place the metric ruler across the zone of inhibition, at the widest diameter, and measure
from one edge of the zone to the other edge.
2. If there is NO zone at all, report it as 0---even though the disc itself is around 7 mm.
3. Zone diameter is reported in millimeters, looked up on the chart, and result reported as
sensitive, resistant, or intermediate.
a. Resistant - indicates that clinical efficacy has not been reliable in treatment
studies.
b. Intermediate - implies clinical applicability in body sites where the drug is
physiologically concentrated or when a high dosage of the drug can be used.
c. Susceptible - implies that an infection due to the organism may be treated with
the concentration of antimicrobial agent used, unless otherwise contraindicated.

Results
(R) Resistant
(I) Intermediate
(S) Susceptible

Antimicrobial Agent E. coli

Amoxicillin (AMC) R
Cephalothin (CF) S
Chloramphenicol (C) S
Ciprofloxacin (CIP) S
Clindamycin (CC) R
Erythromycin (E) R
Oxacillin (OX R
Penicillin (P) R
Streptomycin (S) S
Tetracycline (TE I
Tobramycin (TM)) S
Trimethoprim sulfa (SXT) S

Guide Questions

1. How is the Kirby Bauer test standardized?

Kirby Bauer method relies on the inhibition of bacterial growth measured

under standard conditions. The organism to be tested is grown to a specific turbidity in a
standard liquid medium. The size of the zone of inhibition is directly proportional to the
sensitivity of the organism to the antibiotic.  For a given antimicrobial agent, there is an
approximate linear relationship between the logarithm of the minimal inhibitory concentration
and the zone diameter for organisms with reasonably comparable growth rates. Using their own
standardized technique, Bauer et al3 and Sherris and Schoeknecht12 have correlated zone
diameters of multiple antimicrobial agents with M.I.C.’s as determined by tube and agar dilution
methods. From knowledge of the usually obtainable blood and/or urine concentrations resulting
from standard therapeutic regimes, organisms have been described as sensitive, intermediate
or resistant to given antimicrobial agents based on the diameter of inhibition zones.

2. Describe the Kirby-Bauer disk diffusion susceptibility test protocol?

The validity of this carefully standardized technique depends on, for each defined
species, using discs of correct antimicrobial content, an inoculum which gives confluent growth,
and a reliable Mueller Hinton agar. The test method must be followed exactly in every detail.
After incubation at 35° C for 16–18 hours, zone sizes are measured and interpreted using
National Committee for Clinical Laboratory Standards (NCCLS). These are derived from the
correlation which exists between zone sizes and minimum inhibitory concentration (MIC). The
NCCLS Kirby-Bauer technique should only be used for well-evaluated bacterial species. It is not
suitable for bacteria that are slow-growing, need special nutrients, or require CO2 or anaerobic
incubation. The organism to be tested must be incubated overnight in broth and must be
compared with the 0.5 McFarland Turbidity standard. The media used in this test has to be the
Mueller-Hinton (15x150mm) agar because it is an agar that is thoroughly tested for its
composition and its pH level. Also, using this agar ensures that zones of inhibitions can be
reproduced from the same organism, and this agar does not inhibit sulfonamides.  The agar
itself must also only be 4mm deep.  This further ensures standardization and reproducibility.

The size of the inoculated organism must also be standardized (using the MCFarland
0.5 Latex standard with the Wickersham Card. The reasons are because if the size of the
inoculum is too small, the zone of inhibition will be larger than what it is supposed to be (“the
antibiotics will have a distinct advantage”) and if the inoculum is too large, the zone of inhibition
will be smaller.

Using the published Clinical and Laboratory Standards Institute (CLSI) guidelines,

determine the susceptibility or resistance of the organism to each drug tested. For each drug,
indicate on the recording sheet whether the zone size is susceptible (S), intermediate (I), or
resistant (R) based on the interpretation chart An area of clear media where bacteria are not
able to grow surrounds the wafer, which is known as the zone of inhibition. If the observed zone
of inhibition is greater than or equal to the size of the standard zone, the microorganism is
considered to be sensitive to the antibiotic. Conversely, if the observed zone of inhibition is
smaller than the standard size, the microorganism is considered to be resistant. if some isolates
may have a "relative resistance" it is classified as intermediate.
3. Why is Kirby-Bauer test significant in clinical laboratories?

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.  Clinicians can use Kirby-Bauer test results to choose appropriate antibiotics to
combat a particular infection in a patient. Administering antibiotics that specifically target the
particular bacteria that are causing the infection can avoid using broad-spectrum antibiotics,
which target many types of bacteria. Clinicians use KB test results to choose antibiotics effective
against the specific bacteria causing a patient’s infection. Using specifically-targeted antibiotics
helps decrease the frequency of drug-resistant bacteria evolving.

CONCLUSION

After performing the experiment and interpreting the results, it was determined that
Kirby-Bauer testing measures sensitivity of bacteria to antibiotics by culturing bacteria on solid
growth media surrounding sources of drug. It was also determined that this test is important in
clinical laboratories because it can be useful in monitoring antimicrobials and for the selection of
proper antibacterial agents. In this method of antimicrobial susceptibility testing, The organism
to be tested is grown to a specific turbidity in a standard liquid medium. An inoculum from this
culture is spread across the surface of a Mueller-Hinton agar plate to give confluent growth.
Paper discs containing specific concentrations of antibiotics are placed on the agar surface. The
antibiotic in each disc diffuses outward from the disc, and the concentration of the antibiotic
diminishes as the distance from the disc increases. After incubation, the diameter of the zone of
growth inhibition is measured and scored according to the size of the zone and the particular
antibiotic, as sensitive, intermediate, or resistant.

https://www.coursehero.com/file/25640368/Lab-Report-Ex18docx/

http://shs-manual.ucsc.edu/policy/kirby-bauer-antibiotic-sensitivity

https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiol
ogy_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)

https://courses.lumenlearning.com/boundless-microbiology/chapter/measuring-drug-
susceptibility/

https://www.asmscience.org/content/education/protocol/protocol.3189