Antibiotic susceptibility testing Modified Kirby-Bauer method

Antimicrobial susceptibility testing (AST) Antimicrobial sensitivity testing: to detect the most suitable antimicrobial agent against specific microorganism (drug of choice). WHY? Widespread and non-selective antibiotic usage results in a high driving force in the development of antibiotic resistance

Common methods used for AST Agar diffusion based methods: Disc diffusion method (Kirby-Bauer method) Epsilometer test (E Test)

Kirby-Bauer Method = KB testing or disc diffusion antibiotic sensitivity testing is a test which uses antibiotic impregnated discs to test whether particular bacteria are susceptible to specific antibiotics.

Principle When an antibiotic (present in form of disc) is plated in an agar medium, it diffuses in the surrounding area causing possible inhibition of the bacterial growth in this zone. At the end of incubation period, a clear zone called inhibition zone appears around the antibiotic while the rest of plate shows growth turbidity.

Principle

Principle (Cont.)

Possible techniques Seed or surface inoculation Disc impregnation or ready prepared discs: Disc impregnation: 4 tubes with standard antibiotic concentrations Sterile unmarked filter paper discs Ready discs: are readily prepared antibiotic standard discs for direct use

Procedure Surface inoculation 1- Draw 4 equal sectors with a marker pen on the back of plate and mark the center of each (place of the disc). 2- Pour the agar medium at 50°C in the center of plate and Leave to solidify. 3- Using a sterile swab, inoculate the surface of the medium with the test m.o. in three different directions.

Procedure (cont.): 4- Using a flamed forceps, immerse gently each of the sterile unmarked discs in the antibiotic solution; drain the disc well by touching the inner wall of the tube several times to remove excess solution and place each of the antibiotic discs aseptically on the surface of agar in the center of the sector. Press each disc gently to allow good contact with the medium. 5- Invert the plate and incubate at 37°C for 24 hours.

Sterile swab:-

Expected results Diameter in millimeters R B G V

Expected results