Antibiotic
Sensitivity Testing
Modern era in Antibiotics
begins with Fleming.
History of Antibiotic
Discovery
Uses of Antibiotic
Sensitivity Testing
Antibiotic sensitivity test: A
laboratory test which determines how
effective antibiotic therapy is against
a bacterial infections.
Antibiotic sensitivity testing will
control the use of Antibiotics in
clinical practice
Testing will assist the clinicians in the
choice of drugs for the treatment of
infections.
Components of Antibiotic
Sensitivity Testing
[Link] identification of relevant
pathogens in body fluids collected from
patients
2. Sensitivity tests done to determine
the degree of sensitivity or resistance of
pathogens isolated from patient to an
appropriate range of antimicrobial drugs
3. Assay of the concentration of an
administered drug in the blood or body
fluid of patient required to control the
schedule of dosage
Antibiotic Sensitivity
Testing Is Essential of
selection of Antibiotics
Isolation and Identification of
Bacteria precedes the
selection of Antibiotic Testing
Methods
Why Need continues for
testing for Antibiotic
Sensitivity
Bacteria have the
ability to develop
resistance following
repeated or subclinical
(insufficient) doses, so
more advanced
antibiotics and
synthetic
antimicrobials are
continually required to
overcome them
Testing for Antibiotic
sensitivity
The method includes several steps
including obtaining a bacterial
sample; identifying the type of
bacteria in the bacterial sample;
selecting a set of antibiotics based
on the identity of the bacteria in the
bacterial sample; obtaining a control
sample from the bacterial sample;
Testing Antibiotic Susceptibility
Susceptibility (sensitive) – implies the
organism should respond favourable
to therapeutic doses of the drug.
Intermediate – implies some
susceptibility to therapeutic doses.
Resistance – the usual therapeutic
doses would be ineffective.
Sensitivity testing
techniques
1. Diffusion method
Kirby-Bauer method
Stokes diffusion method
2. Dilution technique
Macrodilution broth susceptibility
test
Agar dilution method
Routine Susceptibility Tests
Disk diffusion (Kirby Bauer)
Broth micro-dilution MIC
E test (epsilometer test)
Kirby-Bauer methods
A commonly used method in
basic laboratories
Kirby-Bauer antibiotic testing
(KB testing or disk diffusion
antibiotic sensitivity testing ) is a
test which uses antibiotic-
impregnated wafers to test whether
particular bacteria are susceptible to
specific antibiotics
How to perform Kirby-
Bauer testing
The basics are easy: The bacterium is
swabbed on the agar and the antibiotic
discs are placed on top. The antibiotic
diffuses from the disc into the agar in
decreasing amounts the further it is
away from the disc. If the organism is
killed or inhibited by the concentration
of the antibiotic, there will be NO
growth in the immediate area around
the disc: This is called the
zone of inhibition .
Disk Diffusion
Test
Prepare inoculum
suspension
Select colonies
Mix well
Standardize inoculum
suspension
Swab plate
Remove sample
Incubate overnight
Add disks
Measure Zones
The area of Inhibition is
measured with a Scale
Record the
results for
everyone on
your table
given below
Measure Zones
Staphylococci – oxacillin, vancomycin
Enterococci – vancomycin
Zone Interpretive Criteria
(mm)
Disk
Drug content Res Int Susc
(ug)
cefazolin 30 14 15-17 18
gentamicin 10 12 13-14 15
The disk diffusion methods are
commonly used for routine
testing
The zone of inhibition
guides the right choice of
Antibiotic
Susceptibility Testing
Methods
Incubate plate
18-24 hr, 35 C
Innoculate Place disks Measure and
MH plate on agar plate record zone of
inhibition around
each disk
Disk Diffusion
Susceptibility Testing
Improper agar & disk placement Mueller Hinton agar &
good disk placement
Use Mueller Hinton agar
Stokes’ Method
In original Stokes’
method the inoculum
of the control strain is
evenly spread over
the upper and lower
thirds of a plate and
that of the test strain
over the central
third;uninoculated
gaps 2 – 3 mm wide
are left to test from
the control areas.
Control strains
in Stokes’ Method
The Microbiology laboratories should
always practice to test the quality control
of their work with the use of standard
strains in Stokes method with following
standard strains and results are
compared
1 Escherichia coli NCTC 10418
2 Pseudomonas aeruginosa NCTC 10662
3 Staphylococcus aureus NCTC 6571
Stokes Method for
Antibiotic Sensitivity
testing
In the Stokes controlled
sensitivity test, a control
organism is inoculated on
part of a plate and the test
organism is plated on the
remainder. Disks are
placed at the interface and
the zones of inhibition are
compared. The use of a
sensitive control shows
that the antibiotic is active,
so that if the test organism
grows up to the disk it may
safely be assumed that the
test organism is resistant
to that drug
The strips with multiple
Antibiotics can be tested in
one go
Other methods of Antibiotic
susceptibility testing
Other methods to test
antimicrobial susceptibility
include the Stokes method, E-
test (also based on antibiotic
diffusion). Agar and Broth
dilution methods for Minimum
Inhibitory Concentration
determination.
Testing Minimum Inhibitory
Concentration
In
alternative measure of
susceptibility is to determine the
Minimum Inhibitory
Concentration (MIC) and the
Minimum Bactericidal
Concentration (MBC) of a drug. A
series of broths are mixed with
serially diluted antibiotic solutions
and a standard inoculum is applied.
What is Minimum
Inhibitory
concentration
Minimum inhibitory concentration
(MIC), in microbiology, is the lowest
concentration of an antimicrobial that
will inhibit the visible growth of a micro
organism after overnight incubation.
Minimum inhibitory concentrations are
important in diagnostic laboratories to
confirm resistance of micro organisms
to an antimicrobial agent and also to
monitor the activity of new
antimicrobial agents.
MIC
Minimal inhibitory concentration
The lowest concentration of
antimicrobial agent that inhibits
the growth of a bacterium
Interpret:
– Susceptible
– Intermediate
– Resistant
Prepare inoculum
suspension
Microdilution MIC tray
Dilute & mix inoculum
suspension
Pour inoculum
into reservoir and
inoculate MIC tray
Incubate
overnight
Inoculate
purity plate
Reflected light
Transmitted light
Examining
purity plate
Read MICs
MICs
- +
0.51
2
4
8
16
32
64
>6 >6
4 4
The Antibiotics are diluted to
various dilution to test the
minimum inhibitory
concentration
MIC test results graphed
Antimicrobial
susceptibility
testing using micro-broth
dilutions
ug/ml
64 32 16 8 4 2
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•
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96 well microtiter plate
What is E Test
Etest is an antimicrobial gradient
technique in which 15 reference MIC
dilutions of an antibiotic have been
repackaged with innovative dry
chemistry technology onto a plastic
strip. The predefined gradient provides
precise and accurate assessment of
antimicrobial activity against both
fastidious and non-fastidious
microorganisms.
The strips are impregnated
with various concentration
of Antibiotics
E = testing on
various isolates
MIC on a
strip
S.
pneumoniae
Penicillin MIC
= 3 g/ml
Antimicrobial Gradient
Testing
E-test®
Read plates
after
recommended
Incubation Read MIC
where elipse
intersects
scale
