Antimicrobial
Susceptibility Testing
by
Staff members of microbiology and
immunology
Faculty of medicine
Helwan university
2020/2021
Objectives:
Identify the meaning and importance of antimicrobial
susceptibility.
Recognize and perform different methods of evaluating
antimicrobial susceptibility :
1- Disc diffusion. 2- Tube dilution. 3- Epsilometer E-test.
Recognize Limitations of antimicrobial susceptibility tests.
Antimicrobials, meaning
what ?
Inhibitors of
bacterial cell
wall synthesis
Inhibitors of
protein
synthesis
Inhibitors of
bacterial nucleic
acid synthesis
What?
Where ?
why?
An in vitro test.
Check the effectiveness of a drug
against a bacterium.
Help to select the best drug that
acts against the bacterium.
Proper
antimicrobial
susceptibility
Effective
Best
treatment
Proper
microbial
identification
Antimicrobial susceptibility tests
Disc diffusion Tube dilution Epsilometer E-
method method test
Minimal inhibitory concentration (MIC) Minimal bactericidal concentration (MBC)
I- Disc diffusion method:
Principle: (Qualitative)
Diffusion
Diffusion of antibiotic
Inhibition of bacterial growth
Creation of inhibition zone
Materials and facilities:
Nutrient broth.
0.5 McFarland turbidity standard:
Sterile swabs:
Cultivation media (Muller Hinton):
Bacteriological loop
Antibiotic discs:
Forceps or antibiotic dispenser
incubator
steps
[Link] preparation
[Link]
[Link] of antibiotic discs
[Link]
[Link]
continued
1- inoculum preparation:
1- inoculum preparation:
Adjust the density to (half
McFarland's).
continued
2- Inoculation:
2- Inoculation:
3- application of antibiotic discs:
continued
4-incubation:
37 °C for 18-24 hours.
5- examination:
zone of inhibition.
continued
• Measure diameter of the inhibition zone by ruler.
According to standard charts organism
susceptibility to antibiotic
Moderately
sensitive Resistant
sensitive
II- Tube dilution methods
Minimal
Minimal inhibitory
bactericidal
concentration
concentration
(MIC)
(MBC)
The minimal antibiotic The minimal antibiotic
concentration that concentration that kills
inhibits bacterial growth bacteria preventing their
and multiplication. growth on plates.
Steps:
Antibiotic preparation (serial dilution)
Inoculum preparation
Reading of MIC
MBC
Pipette
+ 0.75 1.5 3.1 6.2 12.5 25 50
0.2 0.4
control
Drug 50 25 12.5 6.2 3.1 1.5 0.75 No No
µg/ml drug bacteria
0.1 ml
Control
no bacteria 3.1
12.5 6.2
Inoculums
Inoculums constant
preparation amount added Antibiotic
to each tube stock
solution
Antibiotic
serial
dilution
McFarland standards
Plates after Tubes after
incubation incubation
MBC MIC
detection detection
III- Epsilometer E test:
What is the meaning
of Epsilometer?
Diffusion of antibiotic different concentrations
Inhibition of bacterial growth according to antibiotic concentration
Creation of tear shaped inhibition zone with:
Widest part highest antibiotic concentration.
Most narrow part lowest antibiotic concentration.
Limitations of antimicrobial susceptibility
tests?
Limitations of antimicrobial susceptibility:
Measures antimicrobial activity in vitro not in vivo.
Selecting antimicrobial should considers patient general and
clinical conditions.
Activity, action and excretion of the drug should be considered.
Cost and availability of the drug should be considered.
Now !
Let’s revise!
The MIC of the antibiotic in the figure is 0.50 µg/ml
(true – false).
The MIC of the antibiotic in the figure is 0.50 µg/ml
(true – false).
The MIC of the antibiotic in
the figure is
(0.50µg/ml – 1.5µg/ml– 6µg/ml).
The MIC of the antibiotic in the
figure is
(0.50µg/ml – 1.5µg/ml – 6µg/ml).
64µg/ml 32µg/ml 16µg/ml 8µg/ml 4µg/ml 2µg/ml
The MBC of the antibiotic in the figure is
(8µg/ml – 16µg/ml– 32µg/ml).
64µg/ml 32µg/ml 16µg/ml 8µg/ml 4µg/ml 2µg/ml
The MBC of the antibiotic in the figure is
(8µg/ml – 16µg/ml– 32µg/ml).
The MIC of the antibiotic in the
figure is
(4µg/ml – 8µg/ml– 1µg/ml).
The MIC of the antibiotic in the
figure is
(4µg/ml – 8µg/ml– 1µg/ml).
The principle of antimicrobial susceptibility test in the
figure is:
a) Dilution .
b) Diffusion.
c) Dilution and diffusion.
The principle of antimicrobial susceptibility test in the
figure is:
a) Dilution .
b) Diffusion.
c) Dilution and diffusion.