Name: Ylayron, Ma.

Novem Grace Date Performed: November 21, 2017

Ortiz, Karl Andre Date Submitted: November 24, 2017
Calizo, Jenylle Kate
Tolones, Karie Mae
Vargas, Richael
Canieso, Shaina

Section: 3 (T 10:00 AM – 1:00 PM)

Experiment 10
Antibiotic susceptibility testing: Disk Diffusion Assay

I. Objectives

This experiment utilizes specific monitoring techniques to evaluate the

susceptibility of a microbe to different antibiotics; distinguish the range of activity of
an antibiotic; and to recognize and define advantages and limitations of two different
susceptibility testing procedures.

II. Introduction

Antibiotic resistance occurs when an antibiotic has lost its ability to effectively
control or kill bacterial growth; in other words, the bacteria are "resistant" and continue
to multiply in the presence of therapeutic levels of an antibiotic. Bacteria can display
resistance to one or more antibiotics. Determining the bacterial antibiotic resistance is
critically important in the management and control of infectious diseases and pathogens
(web.clark.edu, 2015).
The Kirby-Bauer test, known as the disk-diffusion method, is the most widely
used antibiotic susceptibility test in determining what choice of antibiotics should be
used when treating an infection. This method relies on the inhib ition of bacterial growth
measured under standard conditions. 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. The organism will grow on the agar plate while the antibiotic
“works” to inhibit the growth. 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 (ucsc.edu, 2010).
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 (ucsc.edu, 2010).
 III. Methodology

Materials
3 plates with Mueller-Hinton agar culture of Staphylococcus aureus
antibiotic disks (C30, CFX30, A25, E15) sterile forceps
sterile cotton swab tubes of sterile nutrient broth
inoculating loop McFarland Standard No. 0.5
Culture of Escherichia coli
Culture of Bacillus subtilis
Chloramphenicol, Ampicillin, Amoxicillin and Erythromycin

Using a special slant culture, bacteria were scraped off by a sterilized loop.
These bacteria on the loop where emulsified in a 5mL sterilized broth or saline until the
turbidity is approximately equivalent to that of McFarland No. 0.5 turbidity standard by
comparison to ensure that the inoculum in neither too night or too heavy. This process
is used to inoculate three different bacteria (Staphylococcus aureus, Bacillus subtillis
and Eschericia coli) in separate 5mL sterilized broth.

When desired turbidity is achieved from the three bacteria, a sterilized swab is
dipped into each of the bacterial suspension, excess liquid were ensured to be removed
from the swab, and each of the swab was streaked to the entire surface of different agar
plate. Streaking was repeated twice, in 45° and 90° angle. Each of the plate was divided
into four sections, one section for a different antimicrobial disc that was to be placed.

Forceps were heated, following the aseptic technique, and cooled. The microbial
discs were chloramphenicol, ampicillin, amoxicillin and erythromycin. One microbial disc
was placed at its dedicated section it the inoculated agar, impregnated by pressing the
disc gently using the tip of the forceps. Impregnation is done to regulate the spread of
the microbial disc on the agar.

The plates were labelled properly, inverted, stacked atop each other, placed in a
plastic container and incubated at 35°C for 18 to 24 hours. The results after the
incubation were then recorded.
 IV. Results and Discussion

The procedure used in determining the sensitivity of microorganisms to antibiotics is

the Kirby-Bauer Disc Method also called as agar diffusion method or the disk diffusion
method. After 24 hours of incubation, the plates are examined and the diameter of the
zones of inhibition is measure to the nearest whole centimeter millimeter by the use of
ruler, ignoring the faint growth or tiny colonies which can be detected by very close
scrutiny (Bassiri, 2013). The zone of diameters for the antibiotics used in the experiment
are translated into prefixed susceptible, intermediate or resistant categories through
referring to the Zone of Inhibition Standard Table adapted from Clinical Laboratory
Standard Institute (CLSI) as shown in Table 1.

Table 1.Interpretation of Zones of Inhibition for Kirby-Bauer Antibiotic

Susceptibility Test
Test Cultures (Zone diameters in mm)
Antimicrobial Disc
Potency Resistant Intermediate Susceptible
Agent Code
Amoxycillin –
AMC 20/10 µg
Clavulanic Acid
Enterobacteriaceae ≤ 13 14-17 ≥ 18
Staphylococcus spp. ≤ 19 - ≥ 20
Ampicillin AMP 10 µg
Enterbobacteriaceae
≤ 13 14-16 ≥ 17
and Vibrio cholerae
Staphylococcus spp. ≤ 28 - ≥ 29
Chloramphenicol C 30 µg ≤ 12 13-17 ≥ 18
Enterobacteriaceae,
Staphylococcus
spp., Enterococcus ≤ 12 13-17 ≥ 18
spp., and Vibrio
cholerae
Erythromycin ER 15 µg ≤ 13 14-17 ≥ 18
* Adapted in part from CLSI document M100-S23 (M02-A11) : “Disc diffusion supplemental tablesʼʼ Performance
standards for antimicrobial susceptibility testing. The complete standard may be obtained from the Clinical and
Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA 19807.

From the collected data, the susceptibility of the bacterial samples to the
selected four antibiotics are interpreted in Table 2. Staphylococcus aureus shows that
an intermediate sensitivity to all of the antibiotics used which means that the organism
may be eliminated in body compartment that are easily accessible by the drug for
example the urinary tract, while the same antibiotics may not be adequately effective in
other locations. Dose is required if drugs with intermediate effect against infectious
organism will be used, because this category also serves as buffer zone to avoid
fluctuating test results interpretation (Rodloff et.al, 2008). Escherichia coli on the other
hand, only shows susceptibility to Chloramphenicol, while it developed resistance to the
remaining three antibiotics. The bacterial stain is susceptible to a given antibiotic when
the drug is associated with high therapeutic success, however if the bacteria turns is
resistant to the antibiotic, the drug is said to be associated with high therapeutic failure Formatted: Centered
(Rodloff et. al, 2008). The third bacterial sample is the Bacillus subtilis which exhibits Formatted: Font: (Default) Arial, Bold
sensitivity to Amoxicillin, Chloramphenicol and Erythromycin, but it has a resistance
Formatted: Font: (Default) Arial, Bold
against Ampicillin.
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Table 2. Kirby-Bauer Test Result Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
Formatted: Font: (Default) Arial, Bold
Diameter of
Microorganisms AntibioticAntibiotic Interpretation Formatted: Font: (Default) Arial, Italic
ZOI (mm)
S. aureus AmoxicillinName - I* Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
Ampicillin - I
Chloramphenicol - I Formatted: Font: (Default) Arial, Superscript
Erythromycin - I Formatted: Centered, Position: Horizontal: Center,
E. coli Amoxicillin - R* Relative to: Margin, Vertical: 3.2", Relative to: Page
Ampicillin - R Formatted: Centered, Position: Horizontal: Center,
Chloramphenicol 26 S* Relative to: Margin, Vertical: 3.2", Relative to: Page
Erythromycin 12 R Formatted: Centered, Position: Horizontal: Center,
B. subtilis Amoxicillin 21 S Relative to: Margin, Vertical: 3.2", Relative to: Page
Ampicillin 12 R
Formatted: Font: (Default) Arial, Italic
Chloramphenicol 26 S
Erythromycin 32 S Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
* R (Resistant), I (Intermediate), S (Susceptible)
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Zone of inhibition testing is a fast and inexpensive laboratory tests for Formatted: Centered, Position: Horizontal: Center,
antimicrobial activity and it is a well-suited method in determining the ability of water- Relative to: Margin, Vertical: 3.2", Relative to: Page
soluble antimicrobials to inhibit growth of microorganisms (Lehanie, 2015). However, Formatted: Centered, Position: Horizontal: Center,
there are various conditions that affects the accuracy of the said method. The pH of the Relative to: Margin, Vertical: 3.2", Relative to: Page

medium is an important factor in maintaining the effectiveness of the drugs to be tested. Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
If the pH of the medium is too low, certain drugs such as amino glycoside, macrolides
and quinolones lose their potency, while excessive pH results to smaller or larger zone Formatted: Font: (Default) Arial, Superscript

of inhibition. Selection of antibiotic is based on the type of microorganism to be tested. If
the bacteria is susceptible to the drug then there will be no growth near the disk. The
rate of antibiotic diffusion through the medium is also affects the result, because the rate
is not always constant and it is dependent on the antibiotic concentration. Larger
molecules diffuse slower compared to lower molecular weight compounds. The depth of
the nutrient agar must also be noted since too shallow agar will give false susceptible
results as the antimicrobial compound diffuses further creating larger zone of inhibition.
Too narrow agar depth on the other hand, will produced a false resistant results. The
size of the inoculated microorganisms must also be standardized. Relatively small
inoculum causes the zone of inhibition to be larger than normal, and large inoculum will
Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
Formatted: Font: (Default) Arial, Italic
Formatted: Centered, Position: Horizontal: Center,
Relative to: Margin, Vertical: 3.2", Relative to: Page
Formatted: Centered, Position: Horizontal: Center,
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have a smaller zone of inhibition. Lastly, the presence of other metals such as
excessive thymidine or thymine reverses the inhibitory effects of sulfonamides and
trimethoprim resulting to smaller and few distinct zones of inhibition or there will be no
zones at all. Excess cation concentration will reduced the zone size. On the contrary,
low concentration increases the zone sizes (Ankandhk, 2011).

B. subtilis E. coli

S. aureus
Figure 1. Laboratory Results

V. Conclusion

Based on the experiment, Staphylococcus aureus shows an intermediate

sensitivity to all the antibiotics used. Escherichia coli on the other hand, only shows
susceptibility to Chloramphenicol, while it developed resistance to the remaining three
antibiotics. Bacillus subtilis which exhibits sensitivity to Amoxicillin, Chloramphenicol
and Erythromycin, but it has a resistance against Ampicillin.

Many conditions can affect the accuracy of the test done. Some facors are
the: (a) pH, if the pH of the medium is too low than the desired pH, certain drugs such
as amino glycosides, quinolones and macrolides lose their potency, on the other hand,
antibiotic classes such as tetracyclines appear to have excess activity a lower Ph and
the vice versa happens in the case of the higher pH; (b) Moisture, the presence of
moisture content on the medium can counter act with accuracy of the susceptibility
testing; (c) Effects of medium component, If the media selected for the antibiotic
susceptibility contains excessive amounts of thymine or thymidine compounds, they will
reversibly inhibit the action of certain antimicrobial agents such as trimethoprim groups.
This reversible inhibition yields smaller or less distinct or even no zones and will be
misinterpreted as resistant antibiotics; (d) and amount of organism, The amount of the
organism used for the susceptibility testing is standardized using a turbidity standard.

Kirby-Bauer test is used to identify which bacteria is susceptible to a certain

antibiotic, which is also widely used in the field of medicine, specifically by doctors, to be
able to prescribe the right antibiotic for a certain illness.

References

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