Lab Report 3

Antibiotic Susceptibility Testing

- BIO 223 L -

Student Name: Sara Alabdulkarim

Student ID: 201136
Submission date: October 2, 2021

Abstract:
They invented the Kirby-Bauer test, also known as the disk diffusion method. An
antibiotic susceptibility test can help us determine the best antibiotic to use when
treating an infection or antibiotic resistance. Agar dilution, broth dilution, and disc
diffusion are all options for performing AST. The test is performed by inoculating
the surface of an agar plate with bacteria isolated from a patient's infection, then
applying antibiotic paper disks to the agar, and then incubating the plate. The
antibiotic has been successful if it has stopped the bacteria from growing, and there
will be an area around the disk where the bacteria is no longer visible, this is called
the zone of inhibition. We can compare antibiotic efficacy and monitor antimicrobial
resistance by measuring the diameter of these zones. Finally, we checked for E.coli
and Staph infections. We discovered that E.coli had a diameter of 2cm and
S.pneumonia had a diameter of 2.3cm when using the TN antibiotic. The CD
antibiotic, on the other hand, had no zone of inhibition because E.coli was resistant
to it, and the S.pneumonia had a 3cm diameter.

Introduction:

Objective:
To test an isolated bacteria's susceptibility to a variety of antibiotics.

Materials:
- Stock broth cultures of Escherichia coli
- Sterile cotton swap
- Antibiotic Discs
- Forceps
- Nutrient agar plates
- Flame
Procedure:
 Day (1) Experimental procedure:

1. Label the bottom of the agar plates with group number and date:
a. Discs should be a minimum of 20 mm apart.
b. Discs should not be placed near the edge of the plate

2. Inoculate the plate with your bacterium as follows:

a. Using aseptic technique, wet a swab with the bacterial broth culture. Roll the swab
against the inside of the tube to remove excess liquid.
b. Thoroughly swab the surface of the plate, making sure to cover the entire surface.
c. Do NOT get more culture.
d. Turn the plate approximately 90 degrees and repeat the previous step (2nd swabbing) to
obtain a uniform growth pattern.
e. Do NOT get more culture.
f. Repeat the previous step (3rd swabbing).

3. Discard the swab in a bleach-containing beaker.

4. After 5-10 minutes, place one antibiotic disc onto the surface of the agar, using aseptic
technique as follows:
a. Heat the tips of the forceps by placing them.
b. Cool the forceps by waving them in the air for about 10 seconds.
c. Carefully pick up your test disc with the forceps, and gently place it in the appropriate
spot on the agar surface,
d. To ensure that the disc is flat on the agar, gently push it down with the forceps.
e. Reheat the tips of the forceps to kill any bacteria.
5. Repeat the procedure with the remaining antibiotic discs.
6. Wait until the surface of the plates has completely dried (it may help to leave the lid
slightly open for 3 - 5 minutes).

 Day (2) Observation of K-B test plate:

1. After 24 h, observe the agar plates.

2. If an inhibition present, measure the diameter of the zone of inhibition in mm (see the
two figures below), and record your results. If there is no zone present, record your result as
0 mm.
3. We will then compare the results for the entire class.
 Results:

Figure 2: E.coli bacteria with TN and

Figure 1: Steph bacteria with TN and CD Antibiotics
CD Antibiotics.
Figure 3: Zone of Figure 4: Zone of Figure 5: Zone of
growth inhibition growth inhibition growth inhibition
measurement of steph measurement of measurement of
with CD: 2.5cm steph with TN: 2.3cm E.coli with TN: 2cm
No zone for CD.

Discussion:
In this lab we preformed antibiotic susceptibility testing for antibiotic Tobramycin
(TN) and Clindamycin (CB) on both E.coli and S.pneumonia (staph) bacteria. This
experiment is stretched on two days:
Day 1: Experimental procedure.
Day 2: Observation of test plates.

Day 1:
First, we inoculated two agar plates with both bacteria using the aseptic technique.
We thoroughly swabbed the surface of the plate covering the entire surface. Then
turn the plate 90 degrees and apply the same technique. After 5-10 minutes, we
place an antibiotic disc onto the surface of the agar also using aseptic technique:
First we heat the tips of the forceps then cool them by waving them around. Second
step is to carefully pick up the test disc with the forceps and gently placing both
antibiotic discs on the agar (evenly spaced). To insure that the discs are flat on the
agar, we gently tap the discs down with the forceps.

Day 2:
After 24 hours, we observed the agar plates. We observed that the S.pneumonia
(staph) bacteria showed inhibition for both antibiotics with a 2..5cm diameter for
CD and a 2.3cm diameter for TN. As for E.coli, only TN showed inhibition with a 2cm
diameter and no inhibition for CD.

References:
 Lab manual
 PowerPoint