1. DISK DIFFUSION TESTING
Dr. Samira Fattah
PhD in Medical Bacteriology
College of Health Sciences-HMU

2. Diffusion methods
1. Disk diffusion method
2. Well diffusion method

3. Disk diffusion method(or Kirby–Baur method )
Disk diffusion refers to the diffusion of an antimicrobial agent
of a specified concentration from disks or strips, into the solid
culture medium that has been seeded with the selected
inoculum isolated in a pure culture.

4. Disk diffusion method
• The diffusion of the antimicrobial agent into the seeded culture media results in a gradient
of the antimicrobial.
• When the concentration of the antimicrobial becomes so diluted that it can no longer
inhibit the growth of the test bacterium, the zone of inhibition is demarcated.
• The diameter of this zone of inhibition around the antimicrobial disk is related to
minimum inhibitory concentration (MIC) for that particular bacterium/antimicrobial
combination.
Disk sensitivity test. A – agar; B – antibiotic disc; C – antibiotic diffuses into agar along concentration gradient;
D – bacterial growth on surface of agar after 18 hours of incubation; E – zone (diameter) of inhibition.

5. Disk diffusion method
The diameter of the zone depends on a number of factors including:
1. the quantity of antibiotic within the disk
2. the degree of susceptibility of the bacteria to the antibiotic
3. The depth (in mm) of the agar plate
4. The concentration of bacteria in the inoculum.

6. Disk diffusion method
Factors that affecting results of the sensitivity test:
1. The pH : agar medium should have a pH between 7.2 and 7.4 at room temperature, If the
pH is too low, certain drugs will appear to lose potency (e.g., aminoglycosides,
quinolones, and macrolides), while other agents may appear to have excessive activity
(e.g., tetracyclines).
2. Moisture : The surface should be moist, but no droplets of moisture should be apparent
on the surface of the medium or on the Petri dish covers when the plates are inoculated.

7. Disk diffusion method
Factors that affecting results of the sensitivity test:
3. Inoculum density : Usually optimal results were obtained with an inoculum size that
produced by comparing with McFarland standard .
4. Timing of disc application : If the plates, after being seeded with the test strain, are left
at room temperature for periods longer than the standard time, multiplication of the
inoculum may take place before the discs are applied. This causes a reduction in the zone
diameter and may result in a susceptible strain being reported as resistant.

8. Disk diffusion method
Factors that affecting results of the sensitivity test:
5. Temperature of incubation : Susceptibility tests are normally incubated at 35-37 °C for
optimal growth. If the temperature is lowered, the time required for effective growth is
extended and larger zones result. At higher temperatures, the entire culture appears to be
susceptible.
6. Incubation Time : Most techniques adopt an incubation period of between 16 and 18
hours.
7. Spacing of the antibiotic discs : If larger numbers of antibiotics have to be tested, two
plates, or one 14- cm diameter plate, is to be preferred.

9. Disk diffusion method
Materials Required
• Mueller- Hinton agar
• Antibiotic discs
• Cotton swabs
• Petri dishes
• 0.5 McFarland Turbidity standard
• Pure bacterial culture (24 hrs)
• Forceps
• Metric ruler

10. Procedure:
1. Take several isolated colonies from 24 hours old culture of bacteria to be tested
and immerse the loop in 0.85% saline then compare the turbidity of the
inoculated saline tube with 0.5 McFarland standard.
2. Place a sterile cotton swab in the bacterial suspension and remove the excess
fluid by pressing and rotating the cotton against the inside of the tube above the
fluid level.
3. The swab is streaked in three directions over the surface of the Mueller-Hinton
agar to obtain uniform growth.
4. Allow the plates to dry for five minutes.
5. Using sterile forceps or a suitable disk dispenser, place paper disks on the
surface agar plates at equal distance.
6. Incubate the plates upside down at 37oC for 24 hours.
7. Following overnight incubation, measure the diameter of the zone of inhibition
in millimeter (mm) around each disk.

12. Result and Interpretation
• use a metric ruler to measure the zone of inhibition and include the
diameter of the disk in the measurement.
• Compare the result with CLSI guidelines to report the result.
• The results are reported as Susceptible (S), Intermediate (I), or
Resistant (R).

13. Advantages
• This test is used in determining the proper antibiotics to treat an
infection.
• It doesn’t require special equipment to perform and can be interpreted
by all medical personnel.
• It costs less to perform this test.
Limitations
• Not all slow or fastidious organisms can be tested accurately.
• It is not considered a gold standard test as it is only for screening the
susceptibility pattern of the organisms according to the CLSI
guideline.

14. Disk Diffusion Troubleshooting Guide
- Zones too small……….. Inoculum too heavy
Agar too thick
Disk expired or inactive
Inoculated plates left too long prior to application of disks
Wrong medium for organism
- Zones too large……......... Inoculum too light
Agar too thin
Poor growth (too fastidious, wrong media, not fresh isolate)
- Colonies within zone………Mixed population
- Deformation of zone………. Disks too close to each other