ANTI-MICROBIAL

SUSCEPTIBILITY
TESTING
OBBOH EVANS KOFI
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Definition of terms
.
• Microbiological culture sensitivity tests
It is a method of multiplying microbial organisms in a
predetermined culture media with favorable conditions and then
further testing to check the sensitivity of organism towards the
drugs.

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Purpose of culture sensitivity test

To guide the clinician in selecting the best drug for an individual
patient.
 To control the use of inappropriate drug in clinical practice.
 To accumulate epidemiological information on the resistance of
microorganism.
 To reveal the changing trends in the local isolates.
To overcome the microbial drug resistance
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 Antimicrobial resistance

Antibiotics are powerful drugs and as such carry a wide range of

adverse effects.
Once they are unnecessarily used ,patients experience no benefits
while being still susceptible to the side effects
Antibiotics disrupt the composition of the bacteria leading to
adaptation and resistance.
This makes it a pressing public health problem.
Antibiotic resistance occurs when bacteria evolve to evade the 4
 Methods of Microbiological Sens.
Testing
1. DILUTION METHODS
2. DISC-DIFFUSION METHOD
3. E-TEST
4. AUTOMATED METHODS
5. MECHANISM-SPECIFIC TESTS
6. GENOTYPIC METHODS

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 How the method is selected

the intended degree of accuracy,

convenience, urgency,
bioavailability of sources,
availability of expertise
cost

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 Steps in analysis

 The sample of infective material is spread onto a plate of nutrient agar to

grow and multiply the bacteria.
The bacteria will form colonies that will be exposed to different
antibiotics.
• With a sufficient population of bacteria grown on the plate, the scientist
will perform two main operations:

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1. Identify the species of bacteria
This is done with various techniques, including examination
of characteristics-color, texture, growth pattern, etc.
Gram staining, microscopic examination,
Bacterial species commonly isolated depends on the
location and the cause of the infection.

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2. Determine the bacterial populations sensitivity to a range of antibiotics
This can be done by impregnating the agar with various types of
antibiotics onto the bacterial lawn.

The bacteria are allowed to incubate for a specified days and then later
examined .

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Disc-diffusion method

• 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.

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Procedure

Prepare the inoculum from the initial culture plate by

touching with a loop the tops of 3-5 similar colonies of
the organism to be tested.
Transfer this into a tube of saline
Compare the tube with the 0.5 Macfarland turbidity
standard and adjust the turbidity of the suspension to
that of the standard.

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• Inoculate the plates by dipping a sterile swab into the suspension.
• Streak the swab all over the surface of the medium three times rotating
the plate through an angle of 60 after each application
• Finally pass the swab round the edge of the agar surface

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Procedure contd.

Leave the inoculum to dry for a few minutes (3-5) at room

temperature with the lid closed.
Then, using a dispenser or a pair of sterile forceps with a
template , antibiotic-impregnated disks are placed onto the agar
surface.

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Procedure contd.

As the bacteria on the lawn grow, they are inhibited to varying
degrees by the antibiotic diffusing from the disk.
 The test antibiotic immediately begins to diffuse outward from
the discs.
The plates should be placed within an incubator at 35 within 30
minutes of preparation.

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Procedure contd.

After overnight incubation the diameter of each zone(including the

diameter of the disk should be recorded.
The results should be interpreted according to the antimicrobial
susceptibility interpretation chart.
 Place the metric ruler across the zone of inhibition, at the widest
diameter, and measure from one edge of the zone to the other edge.
Holding the plate up to the light might help .

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The disc diameter will actually be part of that number. If there is
NO zone at all, report it as 0 even though the disc itself is around
7 mm.
 Zone diameter is reported in mm, looked up on the chart, and
result reported as S (sensitive), R (resistant), or I (intermediate).

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 Results for a sensitivity analysis

• The colonies show up as: Susceptible and/or Resistant and/or

Intermediate.
Susceptible: in this case, a clear, circular zone of inhibition- will
appear around the antibiotic disc, indicating an absence of bacteria.

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Resistant: in this case, the filter Paper will have no discernable plaque
around it.
Intermediate: a somewhat cloudy plaque indicates that not all the
bacteria in the area around the disc have been killed.

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Application

To check whether an infection will respond therapeutically to a

particular antibiotic treatment.
This may be utilized as guidelines for chemotherapy, or an
indicator of spread of resistance.

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