Disk Diffusion Testing - Microbiology - Lecture Slides, Slides of Microbiology

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Kirby-Bauer

Disk Diffusion Testing

skill based learning

Principles of susceptibility testing

Kirby-Bauer antibiotic testing

• Kirby-Bauer antibiotic testing ( KB testing or disk

diffusion antibiotic sensitivity testing ) is a test which

uses antibiotic-impregnated paper disks to test

whether particular bacteria are susceptible to

specific antibiotics. A known quantity of bacteria are

grown on agar plates in the presence of thin filter

paper discs containing relevant antibiotics. If the

bacteria are susceptible to a particular antibiotic, an

area of clearing surrounds the wafer where bacteria

are not capable of growing (called a zone of

inhibition).

Agar disk diffusion method

• Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.
• Antibiotic storage -20 o^ C minimum

disks temperature

• Inoculum McFarland 0.5 (10 8 bacteria/mL)
• Incubator temperature 35 o^ C
• atmosphere ambient air

Aseptic transfer of colonies for propagation of

bacteria

Select at least 4 to 5 well- isolated colonies of the same morphological type from an agar plate. Touch the top of each colony with a wire loop and transfer the growth to a tube containing 4 to 5 mL of a suitable broth medium, such as tryptic-soy broth. Allow the broth culture to incubate at 35°C until it achieves or exceeds the turbidity of 0. McFarland standard.

Inoculate the plate with uniformity

• Within 15 minutes after adjusting the turbidity of the inoculum suspension, dip a sterile non-toxic swab on an applicator into the adjusted suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab.

Procedure

• Within 15 minutes of adjusting the turbidity
  • dip a sterile cotton swab into the sample
  • streak a lawn of bacteria on Mueller-Hinton agar

 Leave the lid agar for 3-5 minutes (no more than 15

minutes) to allow plate to dry

Be cautious

• The agar medium should have pH 7.2 to 7.4 at room temperature. The surface should be moist but without droplet of moisture. The antibiotic disks should be maintained at 8°C or lower or freeze at -14°C or below until needed, according to the manufacturer's recom- mendations. Allow the disks to warm to room temperature before use. Don't use expired disks.

Procedure

• Apply antibiotic impregnated disks on the bacterial lawn
  • Important: where the disk drops is where it stays
• Incubate for 16-18 hours at 33 ± 2oC unless otherwise instructed

Do not load the plates with too many

antibiotic disks

• Place the appropriate disks evenly (no closer than 24 mm from center to center) on the surface of the agar plate either by using a sterile forceps or the dispensing apparatus. No more than 12 disks should be placed on one 150 mm plate or more than 5 disks on a 100 mm plate. A disk is not to be moved once it has come in contact with the agar surface since some of the compound diffuses almost instantaneously.

Interpretation

The main concept is the “clinical categorisation"

• Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints
• c and C are the minor and major breakpoints

Susceptible Intermediate Resistant

MIC < c ≤ MIC < C ≤ MIC

Understanding breakpoints

Words of laboratory specialists

• It is not possible to work alone
• Breakpoints are the expression of a consensus among

the scientific community at a given time in a country

Breakpoints are determined using two approaches

• Pharmacological concept
• Epidemiological concept

Follow the guidelines for measuring

zones of resistance and susceptibility

• Interpret the zone sizes by referring to the manufacturer provided standard table and report the organism to be either susceptible, intermediate, or resistant. Never compare the zone sizes of two different antibiotics and judge their effectiveness accordingly.

Results

• Measure the diameters of the zone of inhibition
• Interpret the results as “resistant” or “susceptible” according to the guideline provided by the CLSI - Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination