Antibiotic Sensitivity Tests: Roba Attar Dr. Nisreen Tashkandi

Antibiotic Sensitivity Tests
Roba Attar
Dr. Nisreen Tashkandi
A test done to check the
effectiveness of a drug against a
bacterium and to select the best
drug that acts against the
bacterium.
Antibiotic
Sensitivity
• Right Drug Testing
• Right
Microbe
• Right Cure
Purposes
 To guide the clinician in selecting the best
antibiotic agent for an individual patient.
 To control the use of inappropriate
antibiotics in clinical practice.

 To accumulate epidemiological information on the resistance
of microorganisms of public health importance within the
community.
 To reveal the changing trends in the local
isolates.
 Bacteria have the ability to develop resistance
following repeated or subclinical (insufficient) doses, so more
advanced antibiotics and synthetic antibiotics are continually
required to overcome them.
AST is essential for the selection of
the appropriate antibiotic
K Hari Krishnan
Tirunelveli Medical College
Types
Qualitative
 For the testing of isolates from “healthy” patients with intact immune
defenses.
 For less serious infections such as uncomplicated urinary
tract infections.
Quantitative
 In the treatment of serious infections such as endocarditis
or osteomyelitis.
 For infections in high-risk patient groups such as
immunocompromised patients (e.g.. transplant
patients).
 Those who are critically ill.
Antibiotic Sensitivity
Tests
Diffusion &
Diffusion Dilution
Dilution
Kirby-Bauer Stokes
Tube Dilution Agar Dilution E-Test
Method Method
Qualitative Methods Quantitative Methods

Antibiotics for routine testing
Enterobacteriaceae
Pseudomonas
Staphylococcus Blood &
Intestinal Urinary aeruginosa
Tissues
Benzylpenicillin Ampicillin Sulfonamides Ampicillin Piperacillin

Oxacillin Chloramphenico Trimethoprim Chloramphenic Gentamycin
Erythromycin l Cotrimoxazole ol Tobramycin
Tetracycline Cotrimoxazole Ampicillin Cotrimoxazole
Drugs
Chloramphenicol Tetracycline Nitrofurantoin Tetracycline

Tetracycline Cefalotin
Gentamycin
Diffusion methods
Disk Diffusion Method
Principle
 A paper disk with a defined amount of antibiotic is used to
generate a dynamically changing gradient of antibiotic
concentrations in the agar in the vicinity of the disk.
The basics
The antibiotic contained in a reservoir

is allowed to diffuse out into the
medium and interact in a plate freshly
seeded with the test organisms.
 Inhibition zone edge is formed at the critical time where a
particular concentration of the antibiotic is just able to inhibit
the organism.
INHIBITION
ZONE EDGE
Kirby-Bauer
Method
MATERI
ALS
Mueller-
Hinton Agar
Antibiotic
Disks
Turbidity
Standard
Swabs
Mueller-Hinton Agar
 Medium containing beef infusion,
peptone, and starch.
 Used primarily for the disk-diffusion
method.
Robust red algae (Solieria robusta)

Source of Agar
Mueller-Hinton Agar
 Cool the medium to 45–50 ⁰C and pour into the plates. Allow
to set on a level surface, to a depth of approximately 4 mm.
 A 9-cm plate requires approximately 25 ml of medium.
 When the agar has solidified, dry the plates for 10–30
minutes at 35 ⁰C by placing them in the upright position in the
incubator with the lids tilted.
 If it is not to be used immediately, the agar medium can be stored
in a refrigerator (2 to 8C) for 2 weeks.
Antibiotic Disks
 Any commercially available discs with the proper diameter
and potency can be used.
 Stocks of antibiotic discs can be stored at
-20 ⁰C for 1 month.
 On removal from the refrigerator, the containers should be left
at room temperature for about 1 hour to allow the temperature
to equilibrate.
Antibiotic Disks
Turbidity Standard
 Prepared by pouring 0.6 ml of a 1% (10
g/l) solution of barium chloride
dihydrate into a 100-ml graduated
cylinder, and filling to 100ml with 1%
(10 ml/l) sulfuric acid.
Cotton Swabs
 A supply of cotton wool swabs on wooden applicator sticks
should be prepared.
 They can be sterilized in tins, culture tubes, or on paper,
either in the autoclave or by dry heat.
Cotton Swabs
Procedure
Application of Antibiotic Discs
Incubation
At 35⁰C for 16-18 hours
Measurement of inhibition zone diameter

Kirby-Bauer Method
Procedure
1. To prepare the inoculum from the primary culture plate,
touch with a loop the tops of each of 3–5 colonies, of
similar appearance, of the organism to be tested.
2. Transfer this growth to a tube of saline.
3. Compare the tube with
the turbidity standard and
adjust the density of the
test suspension to that of
the standard by adding
more bacteria or more
sterile saline.
4. Inoculate the plates by dipping a sterile swab into the
inoculum.
Remove excess inoculum by pressing and rotating

the swab firmly against the side of the tube above the
level of the liquid.
K Hari
5. Streak the swab all over the surface of the medium three
times, rotating the plate through an angle of 60⁰ after each
application.
6. Finally, pass the swab round the edge of the agar surface.
K Hari Krishnan
Tirunelveli Medical College
7. Leave the inoculum to dry for a few minutes at room
temperature with the lid closed.
8. The antibiotic discs may be placed on the inoculated plates
using
 sterile forceps.
 a template.
 a sterile needle tip.
 antibiotic disc dispenser.
Incubation

!
 A maximum of seven discs can be placed on a 9–10 cm
plate.
 Six discs may be spaced evenly, approximately 15 mm from
the edge of the plate, and 1 disc placed in the centre of the
plate.
 The plates should be placed in an incubator within 30

minutes of preparation.
 Incubate the plate at 35⁰C – or + 2.
 Disks should not be moved after diffusion.
Strips of multiple antibiotics can
be tested in one go
Incubation

Measurement of diameter
 Using a ruler
 on the under-surface of the
plate containing transparent
medium.
 Using a pair of calipers

 on the plate containing
opaque medium.
Measurement of diameter
 Using automated zone readers
 BIOMIC
 Aura
 Protozone
Interpretation of results
Using a template
 Standard templates are available for each antibiotic.
Using a template
 Result interpretation
 Susceptible
When the edge of the zone of inhibition is OUTSIDE the black
circle.
 Resistant
When there is no zone, or when it lies WITHIN the white circle.
 Intermediate
When the edge of the zone of inhibition lies ON the black circle.
Interpretative chart of zone sizes
Diameter of zone inhibition (mm)
Antibiotic
Resistant Intermediate Susceptible
Tetracycline <14 15-18 >19
Chloramphenicol <12 13-17 >18
Cotrimoxazole <10 11-15 ≥16
Nitrofurantoin <14 15-16 >17
Erythromycin <13 14-22 >23
Gentamycin <12 13-14 >15

 Susceptible
Definitions
 An organism is called susceptible to an antibiotic when the infection
caused by it is likely to respond to treatment with this
antibiotic, at the recommended dosage.
 Resistant
 An organism is called resistant if it is expected not to
respond to a given antibiotic, irrespective of the
dosage and of the location of the infection.
 Intermediate
 Strains that are “moderately susceptible” to an antibiotic that can be
used for treatment at a higher dosage (e.g. b-
lactams) because of its low toxicity.
 Strains that show “intermediate susceptibility” to a more toxic
antibiotic (e.g. aminoglycoside) that cannot be used at a higher
dosage.
Methicillin resistance in Staphylococcus
aureus
Factors influencing
size of zone
 Inoculum density
 Too light inoculum
Inhibition zones will be larger even though the sensitivity of the
organism is unchanged
 Relatively resistant strains may be falsely reported as
susceptible.
 Too heavy inoculum
Inhibition zones will be smaller
 Relatively susceptible strains may then be falsely reported as
resistant.
 Temperature of incubation
 If the temperature is lowered, the time required for effective
growth is extended and larger zones result.

 Potency of antibiotic disks
 If the potency of the drug is reduced owing to
inhibition zone will

deterioration during storage, the
show a corresponding reduction in size.
Primary disk diffusion
 Standardised inoculum is replaced by the pathological
specimen itself, e.g. urine, a positive blood culture, or a
swab of pus.
 Advantage
 Results are obtained 24 hours earlier.
 Disadvantage
 Density of the inoculum cannot be properly controlled.
The results of the primary test should be verified by testing the
isolates subsequently.
AST can be done with
automation
There is a
growing
need for
Automatio
n in
Antibiotic
sensitivity
testing
Antibiotic
Sensitivity Testing
Choose the right drug!

Get faster cure!
Prevent drug resistance!

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Antibiotic Sensitivity Tests

antibiotics in clinical practice.

Qualitative Methods Quantitative Methods

Benzylpenicillin Ampicillin Sulfonamides Ampicillin Piperacillin

Chloramphenicol Tetracycline Nitrofurantoin Tetracycline

The antibiotic contained in a reservoir

Robust red algae (Solieria robusta)

Application of Antibiotic Discs

Measurement of inhibition zone diameter

Remove excess inoculum by pressing and rotating

Measurement of inhibition zone diameter

 The plates should be placed in an incubator within 30

Measurement of inhibition zone diameter

 Using a pair of calipers

Tetracycline <14 15-18 >19

Chloramphenicol <12 13-17 >18

Cotrimoxazole <10 11-15 ≥16

Nitrofurantoin <14 15-16 >17

Erythromycin <13 14-22 >23

Gentamycin <12 13-14 >15

growth is extended and larger zones result.

inhibition zone will

Choose the right drug!

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