Subject Pharm 204 – Pharmaceutical Microbiology with Parasitology

Title The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer
Method)
Activity No No. 7
Introduction Antibiotic susceptibility patterns are called antibiograms. Antibiograms can be
determined by comparing the zone diameter obtained with the known zone diameter
size for susceptibility .
For example, a zone of a certain size indicates susceptibility, zones of a smaller
diameter or no zone at all show that the bacterium is resistant to the antibiotic.
Frequently one will see colonies within the zone of inhibition when the strain is
antibiotic resistant.
Many factors are involved in sensitivity disk testing and must be carefully controlled.
These include size of the inoculum, distribution of the inoculum, 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.
One method that is used to determine antibiotic susceptibility is the sensitivity disk
method of KirbyBauer (named after W. Kirby and A. W. Bauer in 1966).
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 to 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.

Objective(s) At the end of the activity, the student should be able to:
1. 1. Appreciate the scope of antimicrobial activity of selected antibiotics
2. Perform the Kirby-Bauer method for determination of antibiotic
sensitivity
3. Correctly interpret a Kirby-Bauer plate

Materials and • 35°C incubator

Equipment • forceps
• metric rulers
• wax pencil
• 70% ethyl alcohol and beakers
• Bunsen burner
• 4 150 × 15 mm Mueller-Hinton agar plates antibiotic
disk dispensers (BBL or Difco) or assorted individual vials
containing antibiotic disks
• 4 sterile swabs
• 4- to 6-hour tryptic soy broth cultures of Staphylococcus
aureus (ATCC 25903), Escherichia coli (ATCC 11229),
Pseudomonas aeruginosa (ATCC 10145), and Klebsiella
pneumoniae (ATCC e13883)
Procedure/ Instruction
First Period
1. With a wax pencil, mark the lid of each MuellerHinton agar plate with your name, date, and
the name of the bacterium to be inoculated. Each group of students will inoculate the surface of
four Mueller-Hinton plates with S. aureus, E. coli, P. aeruginosa, and K. pneumoniae,
respectively. Use a separate, sterile cotton swab for each bacterium. The swab is immersed in
the culture tube, and the excess culture is squeezed on the inner side of the test tube. If there
are sufficient supplies, you may wish to analyze the antimicrobial sensitivity of microorganisms
from your throat.
2. The swab is then taken and streaked on the surface of the Mueller-Hinton plate three times,
rotating the plate 60° after each streaking. Finally, run the swab around the edge of the agar.
This procedure ensures that the whole surface has been seeded. Allow the culture to dry on the
plate for 5 to 10 minutes at room temperature with the top in place.
3. Dispense the antibiotics onto the plate either with the multiple dispenser or individually with
the single unit dispenser. Make sure that contact is made between the antibiotic disk and the
culture by gently pressing the disk with alcohol-flamed forceps. DO NOT PRESS THE DISK INTO
THE AGAR, AND DO NOT MOVE THE DISK ONCE IT IS PLACED ON THE AGAR
4. Incubate the plates for 16 to 18 hours at 35°C. DO NOT INVERT THE PLATES.
Second Period
1. Measure the zones of inhibition to the nearest mm for each of the antibiotics tested. Record
the results in the report for exercise 43. Use table 43.1 as an aid. For each antibiotic, determine
whether the bacteria are resistant or susceptible.

HINTS AND PRECAUTIONS

(1) If the plate is satisfactorily inoculated, and the inoculum is sufficient, the zones of inhibition
will be uniformly circular and confluent growth should be seen over the entire plate. If you see
isolated colonies on your plate, then the technique performed is less than adequate, and the
procedure should be repeated.
(2) Colonies growing within the zone of inhibition usually result in considering the bacteria drug
resistant.

SAFETY CONSIDERATIONS
Be careful with the Bunsen burner flame. Always handle cultures with care since they may be
potential pathogens. The ethyl alcohol that is used to sterilize the forceps is flammable.

Medical Application
The number of antibiotics (and other antimicrobics) 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. In most clinical laboratories, the antibiogram has been replaced
with molecular techniques.
Data and
Observation
We unwrapped the petri dish
with the culture we had
previously made.

First we diluted We then heated

the sample the tweezers to
medicine to be sterilize them to
used. avoid
unnecessary
contamination.

We used a Using the

puncher to make tweezers, dip the
the small circular paper into the
papers. After diluted medicine.
heating the
tweezers, take
one to be used.

After dipping the paper,

we carefully and gently
placed it on the petri dish.
We also labelled the dish
with non permanent ink
for us to be able to
distinguish which was the
controlled one.

Discussion
The finished
product.
Conclusion
In conclusion the students were able to know the importance of antibacterial
with the help of the Kirby-Bauer Method which was by using antimicrobial
drugs or medicine to test its resistance and observe its zone of inhibition.

After incubating the culture in the petri dish for another few days, we managed
to observe that most of the drugs are still strong against bacteria. The zone of
inhibitions have proved that.

Post Lab
• 1. How can you determine whether the zone of inhibition is due to death or to inhibition of a
bacterium?
Answer: We can determine whether the zone of inhibition is due to death or to
inhibition of a bacterium by swabbing the zone of inhibition and placing it on a new plate, if no
colonies will grow then the bacteria in the zone are dead and if not the zone was only inhibiting
the growth of bacterium.

• 2. What factors must be carefully controlled in the Kirby–Bauer method?

Answer: The factors that must be carefully controlled in the Kirby-Bauer method are;
1. Size of inoculum
2. Distribition of inoculum
3. Incubation period
4. Growth rate of bacterium
5. Depth of agar
6. Diffusion rate of the antibiotic
7. Concentration of antibiotic in the disk.

• 3. In which growth phase is a bacterium most sensitive to an antibiotic?

Answer: The log phase is when the bacterium are most sensitive to antibiotic.

• 4. If the clinical laboratory reports bacterial susceptibility to an antibiotic but the patient is not
responding to it, what could have gone wrong?
Answer: The infection may still be present in a part of the body that the anibiotic is not
Reaching or may have not reached, human error such as wrong patient or wrong culture could
have also contributed to the situation.

• 5. What are the similarities and differences in response to plates with gram-positive and gram-
negative bacteria? Between enterics and nonenterics?
Answer: The only similarities that gram positive and negative bacteria are their layers or
S layers.
Gram positive bacteria have thinner outer membrane and so they are highly
susceptible to antibiotics while gram negative bacteria have largely impermeable cell walls
which excludes certain drugs and antibiotics from penetrating the cell.
 Enteric and nonenteric, both being bacteria don’t have a lot in common. Enteric bacteria
generally do not cause diseases but can be pathogenic under certain conditions and can even
contribute to normal function and nutrition while non-enteric bacteria are pathogenic and can
cause diseases which do not benefit the infected host.

• 6. What is the difference between an antibiotic and an antimicrobic?

Answer: Antibiotics kill and stop growth of bacteria andantimicrobials acts against a wide
variety of organisms.

• 7. What are some reasons bacteria are becoming more resistant to antibiotics?
Answer: Some reasons include;
1. A patient not taking all of the antibiotics to destroy the bacteria completely
therefore allowing it to repair itself and become resistant.
2. A patient not completing his or her medication regime.
3. A patient forgetting to take his or her medicine.