Al-Manara College of Medical Sciences

Department of Pharmacy
2nd Stage // 1st Semester
Practical Medical Microbiology // 5th Lab
The lecturer // Marwa A. Hassan

Antibiotic Sensitivity Testing

Antibiotic: a substance produced by various species of living microorganisms.

Antibiotic sensitivity test: a test done to check the effectiveness of a drug against a
bacterium and to select the best drug that acts against the bacterial infections.

The purposes of sensitivity testing:

1. To guide the clinician in selecting the best antibiotic agent for an individual patient.
2. To control the use of inappropriate antibiotic in clinical practice.
3. To accumulate epidemiological information on the resistance of microorganisms of
public health importance within the community.
4. To reveal the changing trends in the local isolates.

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.

Mueller-Hinton agar is considered the best for routine susceptibility testing of

nonfastidious bacteria for the following reasons:
- It shows acceptable batch-to-batch reproducibility for susceptibility testing.
- It is low in sulfonamides, trimethoprim, and tetracycline inhibitors.
- It gives satisfactory growth of most nonfastidious pathogens.

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- A large body of data and experience has been collected concerning susceptibility tests
performed with this medium.

Kirby-Bauer disc diffusion method

- Procedure
1. To prepare the inoculum from the primary culture plate, touch with a loop the top of
each 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.

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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.

5. Streak the swab all over the surface of the medium three times, rotating the plate
through an angle of 60⁰ after each application.

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6. Leave the inoculum to dry for a few minutes before applying the antibiotic discs, by
using 100 mm diameter petridish, seven disc may be applied (using sterile forceps),
one in centre and six in periphery.

7. The plates are then incubated at 37C⁰ for 16-18 hrs.

8. The zones of complete growth inhibition around each of the disc is included in this
measured.
9. The diameter of disc is included in this measurement.
10. The interpretation of zone size into sensitive, intermediate or resistant is based on
interpretation chart.
- Sensitive: 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.

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11.Measurement of diameter using a ruler (A) or a pair of calipers (B) on the under-
surface of the plate containing transparent medium or using automated zone readers
(C).

(A) (B)

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 (C)

12. The diameter of inhibition zone is measured using a ruler or a pair of calipers.
- This diameter is interpreted according to the critical diameters.
13. Control strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa
etc. should be tested each time when a new batch of agar or discs is used.