Name: Dominique Sofitia R.

Ayson Section: BSMT 3-1

Student No: 19-1-01339 Date: 08-10-21

Worksheet #8
Antimicrobial Susceptibility Testing
Objectives:
1. Explain the rationale behind the performance of antimicrobial susceptibility tests.
2. Describe the method for selection of specific drugs in testing and reporting.
3. Define minimal inhibitory concentration (MIC) and the methods used for
determination of MICs.
4. Explain how zone interpretive criteria used with the disk diffusion test are established.

1. Give the importance of Antimicrobial Susceptibility testing.

 Its importance is that the results from the susceptibility testing helps to determine which
drugs are likely to be most effective in treating a person’s infection. It aids in the
evaluation of treatment services provided by hospitals, clinics and national programs for
control and prevention of infectious diseases. Lastly, it is the monitor of resistance
patterns due to the mutations in bacterial DNA.
 2. Illustrate and discuss all the important steps in performing Kirby Bauer Test.
Put on the gloves. Next label the bottom of the plates.
(you can write your initials, date, and the test)

Take out a sterile swab. It is important not to

contaminate the cotton tip.

Draw a black dot next to the tip. This will help to locate
which side of the cotton swab is inoculated with
bacteria.

Find a single colony to pick. Make sure the black dot is

visible when picking the colony.
 Inoculate a lawn on the blank plate by spreading the
bacteria evenly. It is important to cover the whole plate.
Spread the bacteria again in the perpendicular direction.

Discard the bacteria covered swab in a waste container.

Using the marker, divide the plate in 6 to help evenly

place the antibiotic discs.

Use the tweezers to get 1 antibiotic disc and place in its

own section of the plate.
Gently tap the disc to stick to the agar. The letters T and
E are the abbreviation of the antibiotics full name
(tetracycline) while the numbers signify the
concentration of the antibiotic on the disc (30) for
micrograms.

Reminder:
When placing the discs use the lid to shield the plate
from contamination. If the disc falls out of the tweezers
leave it where it falls. Perform it 5 more times for each
of the antibiotic discs.

Check labels on the discs to make sure that they are all
different.

Place the plated upside down in a 37 degrees Celsius

incubator for 24 to 48 hours or incubate as instructed by
the instructor (teacher).

See the results: after the bacteria have grown, it shows

clearing around the antibiotic discs. It is called the
Zones of inhibition. Wherein the bacteria are only
susceptible to some kinds of antibiotics. Remember:
Antibiotic is most concentrated near the disc.

The concentration to kill the bacteria is by measuring

the diameter of the clearing to determine how
susceptible the bacteria is.
 Draw a line with a marker to measure. Make sure to
measure it in millimeters. Use reference table to
determine whether the bacteria are susceptible, resistant,
or intermediate.

Reminder:
If it is too close to the edge to measure the diameter,
measure the radius by taking the radius and double it to
get the diameter. (diameter=2 x radius)

3. What are the different classification of Antibiotics? Give their mechanism of

action and specific examples of antibiotics and organisms they are indicated.
Classification Mechanism of Action Antibiotic Example Organism
Aminoglycosides Inhibit protein synthesis Gentamicin Enterobacteriaceae
(30s)
Penicillin Inhibit cell wall Penicillin G Strep. pyogenes
synthesis (Grp.A)
Rifampin Inhibit RNA synthesis Rifampin Staphylococcus
Tetracyclines Inhibit protein synthesis Tetracycline Spirochetes
(30s)
Macrolides Inhibit protein synthesis Erythromycin Haemophilus
(50s) influenzae
Cephalosporins Inhibit cell wall Ceftriaxone Staphylococcus aureus
synthesis
Chloramphenicol Inhibit protein synthesis Chloramphenicol Haemophilus
(50s) influenzae
Bacitracin Inhibit cell wall Bacitracin Topical Gram-positive
synthesis
Polymyxins Destroy cell membrane Polymyxin B Topical Gram-negative

4. Illustrate and describe the following reactions after 24 hours of incubation.

a. Susceptible/Sensitive

Description:
 when the bacterial isolate is inhibited in vitro
by a concentration of antimicrobial that is
associated with a high probability of
therapeutic success.
 likely, but not guaranteed to inhibit the
pathogenic microbe; may
be an appropriate choice for
treatment

b. Intermediate

Description:
 When the bacterial isolate is inhibited
In vitro by an antimicrobial concentration
That is associated with an uncertain
Therapeutic effect.
 may be effective at a higher dosage,
or more frequent dosage, or effective
only in specific body sites
where the antibiotic penetrates
to provide adequate concentrations

c. Resistant
 Description:

 when the bacterial isolate is inhibited

In vitro by an antimicrobial concentration
That is associated with a high
Probability of therapeutic failure.
 not effective in inhibiting the growth of
the organism; may not be an
appropriate choice for
treatment

REFERENCE(S):
https://www.slideshare.net/SaajidaSultaana/antimicrobial-susceptibility-testing-
198411709

Moore, D. W. (2021). Antibiotic Classification & Mechanism - Basic Science - Orthobullets.

Orthobullets.com. https://www.orthobullets.com/basic-science/9059/antibiotic-classification-
and-mechanism

Great Diseases. (2016). ID Laboratory Videos: Antibiotic susceptibility testing [YouTube Video].
In YouTube. https://www.youtube.com/watch?v=Np87w5kCL-4