LAB 9 Antibiotics Susceptibility Tests 2nd 2020-2021

Faculty of Medicine and Health Sciences
General Microbiology Lab (7105404)
Lab-9
Antibiotics Susceptibility Tests
Second Semester 2020-2021
1
Introduction:
Antimicrobial Agents: are chemical substances that either kill or inhibit the growth of microorganisms
without significantly causing harm to the host (patient) cells.
Bacterial pathogens are remarkably capable to develop resistance to antibiotics. In addition, their
antibiotic resistance profiles are not possible to be predicted.
Accordingly, it is important to examine their antibiotics susceptibility profile before initiating the
treatment process.
Antibiotics Susceptibility Testing:
These are methods that can be used to determine the antibiotics susceptibility profile of a bacterial
pathogens, which include:
I- Conventional methods:
1.Broth dilution
2.Agar dilution
3.Disk diffusion
II-Commercial methods:
4.Antibiotic strips
5.Computerized automated systems
I- Broth Dilution Method:
During this method:
 Broth culture tubes (Mueller-Hinton broth) are prepared with a two-fold serial dilutions of a
certain antibiotic.
 Then, the tubes are inoculated with the bacterial pathogen intended to be tested.
 After that, the tubes are incubated for 24h at 37ºC.
 By the end of the incubation period, the tubes are examined for turbidity (which reflects the
growth of the inoculated bacterium).
 The lowest concentration of the used antibiotic capable of preventing the growth of the tested
bacterium (no turbidity is seen in broth) is known as the minimum inhibitory concentration
(MIC) of that antibiotic.
Definitions:
Minimum Inhibitory Concentration (MIC): it is the lowest concentration of an antibiotic that

prevents the growth of the tested-bacterium in vitro.
A bacterium is said to be resistant to a particular antibiotic when its MIC reaches to a certain level
(in vitro) that cannot be reached in the body (or blood) of the patient (in vivo), because, in this case,
this antibiotic will becomes toxic to the patient.
Minimum Bactericidal Concentration (MBC): It is the lowest concentration of an antibiotic that

kills the tested-bacterium in vitro.
The MBC of a tested antibiotic can be determined by:
1- Sub-culturing the broth tubes that show no growth (clear/no turbidity) onto antibiotic free- agar
plates (such as nutrient agar).
2- Then, these plates are incubated for 24h at 37ºC.

3- The plate that shows no growth determines the MBC, which represents the concentration of the
tested antibiotic in the broth tube from which this plate was inoculated.
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II- the Agar Dilution Method:
In this method:
 A set of Mueller-Hinton agar plates (instead of Mueller-Hinton broth) are prepared with a
two-fold serial dilutions of the antibiotic intended to be tested.
Then, the bacterial pathogen that is being examined (prepared at 1.5 × 108 CFU/ml) is
inoculated onto the surface of the agar medium.
After that the plates are inoculated at 37 ºC for 24h.
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Limitation of both the broth and the and agar dilution antibiotics susceptibility testing:
Although the broth and agar dilution method are very accurate in determining both the MIC
and the MBC, however, these methods require a lot of work to be used on a daily- basis in
Medical Microbiology Labs.
Accordingly, it was necessary to develop a more practical and a less-time consuming

method that can give reliable results. i.e. Disk Diffusion method.
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III- The Disk Diffusion Method (The Bauer-Kirby Test) :
This test is commonly used in medical microbiology to determine the antibiotic susceptibility
profile of bacterial pathogens.
To conduct this test:
A suspension of a freshly-cultured bacterium intended to be examined is

prepared (0.5 McFarland =1.5 × 108 CFU/ml)
Then, the bacterium is inoculated uniformly onto the

surface of Mueller-Hinton agar plates using a sterile cotton
swab
After that and by using a

sterilized forceps, commercially-
prepared antibiotic disks are
evenly placed (at least 2.5 cm
apart) on the surface of the
inoculated agar plates (each type
of these disks contains a
standardized amounts of an
antibiotics.
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The plates are then incubated at 35 ºC for 16-18h
During the incubation time, the antibiotic in each

of the placed disks will diffuse in through the agar
medium to cause growth inhibition of susceptible
bacteria. This inhibition appear as clear zone (no
bacterial growth) around the antibiotic disk as
shown in figure.
Reading the Results:
 The diameter of any resulting zones of inhibition

is measured in millimeters (mm) as shown in the
image below.
 The bacterium is classified as susceptible,

moderately susceptible, intermediate, or resistant to
each antibiotic through using a standardized table
usually according to Clinical and Laboratory
Standards Institute (CLSI).
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Commercial Susceptibility Testing Systems
A- The E-test (Epsilometer test):
The MIC is mainly determined by either the broth or
the agar dilution methods. However, it is possible by
the E-test, which a diffusion-based test, to link the
inhibition zone to MIC by using a strip that contains a
gradient amounts of an antibiotic.
Procedure:
The bacterium (0.5 McFarland suspension) is
inoculated uniformly onto the surface of Mueller-Hinton
agar plates using a sterile cotton-swab.
Then, several E-strips, (each of which contains
gradient amount of an antibiotic) are placed on the
surface of agar medium.
Then, the plates are incubated at 37 ºC for 24h
During the incubation time, each of the E-strips creates
an antibiotic gradient in the agar medium.
This will inhibit the growth of susceptible bacteria,
where MIC can be read at the lower intersection between
inhibition zone and the strip.
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B- MIC determination using an agar medium that contains a gradient of an
antibiotic concentrations
Growth patterns on a plate containing an

antibiotic gradient (concentration
decreases from center of the plate to the
periphery) is observed.
The distance from where growth is noted

at the edge of the plate to where growth is
inhibited toward the center of the plate is
measured.
This value is used in a formula to

calculate the MIC of the antimicrobial
agent against each of the bacterial
isolates streaked on the plate. (Courtesy
Spiral Biotech, Inc., Bethesda, Md.)
Source: https://www.americorpshealth.biz/susceptibility-testing/b-cez.html
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Automated Tests:
Computerized-automated tests have been developed for antimicrobial susceptibility

testing.
These tests measure the inhibitory effect of the antimicrobial agents in a liquid medium
by using light scattering to determine growth of the test organism.
Results can be obtained within a few hours
Labs performing very large numbers of susceptibility tests frequently use the automated
methods but the equipment is quite expensive.
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Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Second Semester 2020-2021

During this method:

Minimum Inhibitory Concentration (MIC): it is the lowest concentration of an antibiotic that

Minimum Bactericidal Concentration (MBC): It is the lowest concentration of an antibiotic that

The MBC of a tested antibiotic can be determined by:

2- Then, these plates are incubated for 24h at 37ºC.

Accordingly, it was necessary to develop a more practical and a less-time consuming

To conduct this test:

A suspension of a freshly-cultured bacterium intended to be examined is

Then, the bacterium is inoculated uniformly onto the

After that and by using a

During the incubation time, the antibiotic in each

Reading the Results:

 The diameter of any resulting zones of inhibition

 The bacterium is classified as susceptible,

Growth patterns on a plate containing an

The distance from where growth is noted

This value is used in a formula to

Computerized-automated tests have been developed for antimicrobial susceptibility

Results can be obtained within a few hours

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