Veterinary Epidemiology and Zoonosis

EXERCISE-8: MODIFIED KIRBY - BAUER METHOD

• Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth invitro.
• This ability may be estimated by either the dilution method or the diffusion method.
• The recommended method is the modified Kirby-Bauer method.

Modified Kirby-Bauer method

Materials required

Materials required	Quantity and purpose
Sterile swabs or containers	To collect clinical material or test samples
Nutrient agar or BHI agar	Adequate quantity
Nutrient broth or BHI broth or tryptic soya broth	Adequate quantity
Mueller-Hinton agar	Adequate quantity
Petriplates and distilled water	Adequate quantity
Pressure cooker	To boil and sterilize the medium
Antibiotic discs, disc dispenser or forceps, spirit lamp	Adequate quantity
Incubator	To incubate culture plates

Procedure

• Milk sample can be directly smeared on agar plate with a sterile swab.
• In case of pus swab, heart blood swab or swabs from any organ, the sample should be cultured on nutrient agar or BHI agar.
• After incubation period , pick 3-5 colonies of similar appearance and transfer to BHI broth or tryptic soya broth.
• Incubate the broth cultures at 35±2°C for 2-4 hours for development of light to moderate turbidity.
• Streak the broth culture over the surface of the medium.
• Place the antibiotic discs to the inoculated plates using an antibiotic disc dispenser or sterile forceps.
• The plates should be placed in an incubator at 37°C within 30 minutes of preparation and incubated for 16-18 hours (24 hours for Staphylococci).
• Do not incubate the plates in an atmosphere of carbon dioxide.

Interpretation of result

• After overnight incubation, the diameter in mm of each zone (including the diameter of the disc) should be measured and recorded.
• The results should then be interpreted by comparing with standard tables as susceptible, intermediate and resistant to the antimicrobial agents used.

Figure: Antibiotic discs showing ‘zone of inhibition’ of bacterial growth