Kirby – Bauer disc diffusion method

Aim

To determine the antibiotic sensitivity of the given organism by Kirby – Bauer disc diffusion method.

Principle

Kirby – Bauer disc diffusion method is commonly employed for antibiotic sensitivity test. The test is based on the fact that for a given antibiotic, the size of zone of inhibition is related to MIC                 {Minimum  Inhibitory Concentration }. MIC is referred as the lowest concentration of the antibiotic that exhibits the zone of inhibition on the assay plate. Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacterial isolate

Materials required

·         Sterile forceps

·         Sterile swabs

·         Broth culture of the organism

·         Sterile Mueller-Hinton agar plates

·         Antibiotic discs

 

Procedure

Preparation of plates

Sterile Mueller-Hinton agar [pH 7.3] was prepared and poured into plates [the depth of the medium should be approximately 4mm]. After solidification the plates were dried for 30 minutes in an incubator to remove excess moisture from the surface.

Preparation of inoculum

·         Only clinical isolates are used for  antibiotic sensitivity test.

·         5-6 selected colonies were inoculated into nutrient broth with the help of inoculation loop

·         The broth culture was incubated at 37oC for 2-5 hours.

Inoculation

·         A sterile cotton swab was dipped into the diluted inoculum and rotated the swab inside the wall of the tube to remove excess inoculum

·         The agar surface of the plate was streaked in the three directions by turning the plate 60o angle between each streaking

·         The petriplates were closed with their lids and kept at room temperature for 5-10 minutes to dry the inoculum.

 

Application of disc

The antibiotic discs were removed  from their respective vials with the help of flamed forceps and carefully placed them in the plates, at least 24mm away from the edge . Equal distance of placement is required to avoid the overlapping of the zone surface of the medium. The plates were allowed to stand at room temperature for 30 minutes [Pre diffusion time]

Incubation

The plates were incubated at 37oC for 16 – 18 hours.

Reading the result

The diameter of the zone of inhibition is measured at the end of the incubation period .the zones measured,  only that are showing complete to the nearest millimeters

Result

Eight different antibiotics were used against ……… sp. For sensitive and resistant pattern. Among these only …….. was resistant and …….. were sensitive . ………. showed maximum zone of inhibition[…….. mm] against ………. sp.

 


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