Introduction

The presence of antimicrobial agents at low concentration through leaching or continued

usage may lead to the development of drug-resistant strains and Multiple Antibiotic
Resistance (MAR) in bacteria which may result to resistance transfer to pathogenic bacteria
and reduced efficacy of antibiotic treatment for human and animal diseases. Several studies
have been done to investigate the possible consequences of the used of antimicrobials.

Antibiotic susceptibility pattern has long been utilized as useful epidemiological markers
since it is very simple to perform and the results could be obtained in a ver y short time and
are easy to interpret (Tenover et al., 1995). The existence of bacterial antibiotic resistance
may be as a result of non-clinical used of antibiotics in both animal and humans.

Resistance to antimicrobial compounds can be conferred by innate structural features of

microorganisms such as an impermeable outer membrane that resists penetration of
antibiotics. Gram-negative bacteria have a thick lipopolysaccharide layer that acts as a barrier
to limit diffusion of antibiotic molecules into the cell while gram-positive bacteria
characteristically have lipophilic substances in their cells walls that retards penetrations of
hydrophilic, cationic and antimicrobial compounds. In addition to barriers, microorganisms
may possess a variety of other resistant mechanism. For example, the organism may lack a
transport system necessary for antibiotic uptake or be lacking the biochemical target required
for attachment and proper functioning of the antimicrobial compound (Volk et al., 1996).

The multiple antibiotic resistance index of the isolates is defined as a/b where ‘a’ represents
the number of antibiotics to which the particular isolate was resistant and ‘b’ the number of
antibiotics to which the isolate was exposed to (Krumperman, 1983).

Objectives

To expose student with the knowledge of the Antibiotic Susceptibility Test

Materials:

1. Mueller Hinton Agar

2. Pure culture
3. Sterile cotton swab
4. Antibiotic discs
Methodology:

1. Briefly, organisms were grown at 37ºC in Mueller Hinton broth.

2. Ten microliters of the overnight culture were used to inoculate a fresh
3. Mueller Hinton broth followed by incubation at 37 ºC with shaking until a 0.5
McFarland turbidity standard (biomerieux, France) was obtained.
4. A sterile swab was dipped into this culture and used to inoculate the surface of a fresh
Mueller Hinton agar plates and were allowed to dry for 2 to 5 minutes.
5. The antibiotics discs were spaced out onto the plates and were incubated at 37ºC for
24 hours.
6. The diameter of the clear zone or each antibiotic disc was interpreted as susceptibility
or resistant categories according to the guideline recommended by the National
Committee for Clinical Laboratory Standards (2004).

Observation:

Antibiotics Discs
Types of Culture Streptomycin Nycin Kanamycin

Staphylococcus aureus

Diameter of clear zone: Diameter of clear Diameter of clear

zone: zone:
1.8 cm
1.8 cm 2.0 cm

susceptible susceptible
susceptible

Salmonella sp.

Diameter of clear zone: Diameter of clear Diameter of clear

zone: zone:
1.8 cm
1.8 cm 2.2cm

susceptible susceptible
susceptible
 Diameter of clear zone: Diameter of clear Diameter of clear
zone: zone:
2.0cm
1.8 cm 2.2cm
Vibrio para

susceptible susceptible
susceptible

Discussion:

Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics.

Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic
will be most successful in treating a bacterial infection in vivo.

In the disk-diffusion susceptibility test, disks containing known amounts of an antimicrobial

agent are placed on the surface of an agar plate containing a non selective medium that has
been inoculated with a suspension of a strain. The antimicrobial agent diffuses into the
medium, causing a zone of inhibition of growth of the strain around the disk corresponding to
the susceptibility of the strain to the agent. Bacteria are not able to grow around antibiotics to
which they are sensitive.

Interpretative inhibition zone diameters have been established for susceptibility test results to
permit classification of an isolate as being susceptible, intermediate (or exhibiting decreased
susceptibility), or resistant to an antimicrobial agent.

From the result obtained, we can see that kanamycin inhibit the growth of bacteria the most
followed by streptomycin and nycin. Usually, according to National Committee for Clinical
Laboratory Standards, susceptible categories is interpreted when the clear zone is ≥ 18 mm.
From the result, we can be confirmed that all the bacteria were susceptibled to all 3
antibiotics.
Conclusion:

From this experiment, we learned to perform Antibiotic Susceptibility Test and observe the
result on how certain bacteria sustained to certain antibiotics. We also learned to measure the
diameter of the clear zone and how to classify the isolate as being susceptible, intermediate or
resistant according to National Committee for Clinical Laboratory Standards (2004).

BCS 3224
MEDICAL MICROBIOLOGY

LAB EXERCISE 3
ANTIBIOTIC SUSCEPTIBILITY TEST
 LECTURER: MISS INTAN FARAHA

NAME: FARHANNA MOHAMMED

MATRIC NUM: 870131565028
PROGRAM: ISM INDUSTRIAL BIOTECHNOLOGY