Part 2

(D) Procedures
1. The hands and the workbench were sprayed with ethanol and wiped with a sterile
tissue paper.
2. The Bunsen burner was it up and was set up such that a non-luminous flame was
observed.
3. The 9 agar plates are turned upside down such that the lids of the agar plates are facing
the workbench.
4. 1 sterile cotton swab was dipped into the S. aureus broth and was rolled against the
inner wall of the test tube broth such that any excess S. aureus broth was removed.
5. The cotton swab coated with S. aureus broth was then gently swabbed across the agar
plate in a zigzag pattern up to the edges of the agar plate and in a top-to-bottom fashion
as shown in Diagram 1 to form an even bacterial lawn.

Starting position

Ending position Diagram 1 Illustration of Step 5

6. The agar plate was rotated 60 and Step 5 was repeated as shown in Diagram 2.

Starting position

Diagram 2 Illustration of Step 6

(Step 6 is colored in red for clarity)

Ending position

7. The agar was further rotated 60 and Step 5 was repeated as shown in diagram 3.

Starting position

Diagram 3 Illustration of Step 7

(Step 7 is colored in blue for clarity)

Ending position
8. Steps 4 to 7 were repeated on two other agar plates. The three inoculated agar plates
were labeled with the species of bacterium, name and date of inoculation and the group
number on the side of the base of the Petri dish separately with a permanent marker.
The base of the Petri dish was separated into 3 even portions using a permanent
marker.
9. A forcep was flamed using a Bunsen flame, then was used to pick up 9 different types of
commercial antibiotic discs and put into the 3 agar plates in an even spacing as shown in
Diagram 4. 1 sterile paper disc was put into the center of each agar plate as a control.
The discs were gently pressed to the agar to ensure that the discs were attached to the
agar.

Diagram 4 Placement of commercial

antibiotic discs and sterile paper discs

10. Steps 4 to 10 were repeated with M. luteus and E. coli respectively.

11. Incubate the agar plates in an incubator in an upside-down fashion and in 37 degrees
Celsius for 24 hours.
12. The agar plates were taken out after 24 hours. An electronic caliper was put across the
zone of inhibition at the widest diameter, and was measured from one edge of the zone
to the other edge. The clear zone was defined as the circular area around a paper disks
in which no bacteria was present. The diameter of the clear zone was recorded.

Image 5 Demonstration of
measuring the clear zone
with an electronic caliper
Precautions

Various precautions were implemented in this experiment as shown below.

1. The workbench was sterilized with 75% ethanol solution to reduce the risk of other
bacteria from entering the agar plate and ensure that only the desired species of
bacteria is able to grow on the agar plate.
2. The forceps were sterilized with the Bunsen flame to prevent any antibiotics on the
paper disc from reacting with the bacteria on the forceps.
3. The agar plates were put on the workbench with the lids of the agar plates facing the
bottom of the agar plate to prevent entry of other unwanted species of bacteria into the
agar plate.
4. The hands of the person doing the experiment should be washed before and after the
experiment to reduce the infection of bacteria.
5. The agar plate was smeared entirely with bacteria to allow for an even spreading of
bacteria on the plate for a fairer result.
6. The lengths of the antimicrobial discs were standardized by an antimicrobial disc
dispenser.
7. One new antimicrobial disc dispenser was used for each antimicrobial disc to prevent
the contamination of other antibiotics.
8. The agar plate was brought against the light to be measured such that the clear zone
was much clearer for measurement.
9. The clear zone measurement was repeated for 3 times and taken average to improve
the accuracy of the measurement.
10. The pH value of the agar jelly was kept at pH 7.2 to pH 7.4 at room temperature to
prevent the appearance of false Susceptible or Resistant results.

(E) Results
Zone diameter Rating
Bacteria species Antibiotics
(mm) (R,I,S)
Staphylococcus aureus Chloramphenicol (C 30) 20.20 S
(Gram positive) Erythromycin (E 30) 26.84 S
Kanamycin (K 30) 21.10 S
Neomycin (N30) 16.55 I
Penicillin (P 10) 38.41 S
Streptomycin (S 25) 19.47 S
Tetracycline (TE 30) 30.02 S
Colistin sulphate (CT 25) 9.54 R
Novobiocin (NV 30) 30.19 S

Micrococcus luteus Chloramphenicol (C 30) 8.71 R

(Gram positive to Erythromycin (E 30) 16.01 I
Gram variable) Kanamycin (K 30) 27.55 S
Neomycin (N30) 2.79 S
Penicillin (P 10) 41.66 S
Streptomycin (S 25) 16.16 S
Tetracycline (TE 30) 46.26 S
Colistin sulphate (CT 25) 15.50 S
Novobiocin (NV 30) 41.48 S

Escherichia coli (Gram Chloramphenicol (C 30) 27.75 S

negative) Erythromycin (E 30) 13.16 I
Kanamycin (K 30) 25.24 S
Neomycin (N30) 18.81 S
Penicillin (P 10) 8.63 R
Streptomycin (S 25) 29.66 S
Tetracycline (TE 30) 26.85 S
 Colistin sulphate (CT 25) 14.56 S
Novobiocin (NV 30) 11.10 R
(Legend: S=Susceptible, I=Intermediate, R=Resistant)

Table 6 Significance of zones of inhibition in

Antimicrobial Susceptibility Test

Clear zone diameter of different antibiotic discs in S. aureus bacterial

lawn (Gram positive bacteria)
45.00
38.41
40.00
35.00
Zone diameter (mm)

30.02 30.19
30.00 26.84
25.00 21.10
20.20 19.47
20.00 16.55
15.00
9.54
10.00
5.00
0.00

Legend: Red=Susceptible, Blue=Intermediate, Yellow=Resistant Diagram 7 Clear zone and susceptibility

of S. aureus against various antibiotics

Clear zone diameter of different antibiotic discs in M. luteus bacterial

lawn (Gram positive to Gram variable bacteria)
50.00 46.26
45.00 41.66 41.48
40.00
Zone diameter (mm)

35.00
30.00 27.55
25.00 22.79
20.00 16.01 16.16 15.50
15.00
8.71
10.00
5.00
0.00

Legend: Red-Susceptible, Blue=Intermediate, Yellow=Resistant

 Diagram 8 Clear zone and
susceptibility of M. luteus
against various antibiotics

Clear zone diameter of different antibiotic discs in E. coli bacterial lawn

(Gram negative bacteria)
35.00
29.66
30.00 27.75 26.85
25.24
Zone diameter (mm)

25.00
18.81
20.00
14.56
15.00 13.16
11.10
10.00 8.63

5.00

0.00

Legend: Red=Susceptible, Blue=Intermediate, Yellow=Resistant

Diagram 9 Clear zone and
susceptibility of E. coli
against various antibiotics
According to Diagram 7, S. aureus was found to be susceptible to 7 out of the 9 antibiotics.
S. aureus was found to be intermediate to Neomycin, and resistant to Colistin sulphate.

According to Diagram 8, M. luteus was found to be susceptible to 6 out of the 9 antibiotics.

M. luteus was found to be intermediate to Erythromycin and resistant to Chloramphenicol
and Kanamycin.

According to Diagram 9, E. coli was found to be susceptible to 6 out of the 9 antibiotics. E

coli was found to be intermediate to Erythromycin and resistant to Penicillin and
Novobiocin.

(F) Discussion
The results showed that on the criteria of Gram positivity or negativity, Gram-negative
bacteria generally showed a higher resistance to antibiotics. This may be due to the
presence of an impermeable cell wall. Gram-negative bacteria have an impermeable
lipopolysaccharide and protein-containing outer membrane. This membrane has slowly
evolved over time to become a protective mechanism against antibiotics. Therefore, the
Gram-negative bacteria such as E. coli are found to have a higher resistance against
antibiotics. The clear zones were also smaller when compared to those that have been
putted in the agar solution of other Gram-positive bacteria.
The reason for the appearance of the clear zones is that during incubation, the bacteria
divides and grows in the nutrient agar jelly by binary fission. However, if the bacteria have
any susceptibility to the antibiotic in testing, it will be unable to grow in the areas of
inhibitory antibiotic concentration. The zone diameter of a certain antibiotic and bacteria
combination is inversely related to the minimum inhibitory concentration, which is the
lowest concentration of an antibiotic which prevents the visible growth of a bacterium.

Various sources of error are present in this experiment. Firstly, the number of times of
taking the data was not enough to provide an accurate result. The cotton swabs for
streaking the agar plate were not disposed of immediately after streaking. The commercial
antibiotic discs were not put evenly on the agar plate which lead to the clashing of two clear
zones in the experiment. The density of the bacteria on the agar plate was not standardized,
which would lead to the false recording of resistant if the bacterial density on the agar plate
was too thick. The antibiotic discs were not placed firmly on the agar plate which led to the
sliding of the discs and led to a non-circular clear zone to be formed.

Improvements can be made in this experiment. Firstly, the data could be retaken for at least
five times and then taken average if the time allows to provide more accurate results for the
experiment. The aseptic techniques should be enacted more frequently in order to reduce
the spreading of bacteria to prevent the risk of infection and the contamination of other
agar plates. The position of the antibiotic discs should be accurately marked with a marker
to prevent the clashing of clear zones. A standardized inoculum should be applied to the
agar and the same person should be tasked to streak all the agar plates to reduce the
difference in bacterial density caused by human error. When placing the antimicrobial discs,
the discs must be pressed gently on the agar with the sterilized forceps to ensure that the
discs will not slide and move during incubation.

There are limitations to the antimicrobial susceptibility test (Kirby-Bauer Test). The results
obtained may be at the borderline between two categories or may be unexpected. This will
require further testing in order to obtain the results. Moreover, the Kirby-Bauer Test is not
applicable for usage of viruses, slow growing microorganisms, and certain antibiotic
resistant bacterium, such as penicillin-resistant S. pneumoniae. The chemical properties of
antibiotics also set some limitations to the Kirby-Bauer Test. Some antibiotics have a large
molecular size and mass, such as Vancomycin. This makes them very slow to diffuse in the
agar jelly. Therefore, the clear zone can only reach a short distance, which makes the results
unreliable, as the difference between the different categories is only separated by a small
margin. Moreover, there are different non-standardizable factors which determine the clear
zone diameter such as the solubility of the antibiotic in the agar solution, the thickness of
the agar jelly as well as the concentration of the antibiotic in the disc. This leads to the fact
that the Kirby-Bauer Test can only provide a limited amount of information on the
susceptibility and the resistance to the tested antibiotics. The Kirby-Bauer Test cannot
differentiate between bacteriostatic and bactericidal activities, and the difference in zone
sizes cannot be compared to antibiotic efficiencies, as the antibiotic concentrations in each
disc are different. The Kirby-Bauer Test can only tell whether a bacterium is susceptible or
resistant to a certain antibiotic.
Conclusion
Gram-positive bacteria showed to have a higher susceptibility to antibiotics than Gram-
negative bacteria.