Antimicrobial Activity - Disk Diffusion method ( Kirby- Bauer method)

Disc diffusion method is to determine the antimicrobial activity  or Antibiotic susceptibility of testing organism . This test can be used to check the therapeutic potentials of various sources like., plant, microbes, nanoparticles etc.,

Requirements:

Mueller Hinton agar plate
Sterile cotton swab
Stock solution of plant extract/ other extracts (antimicrobial agent)
Bacterial broth culture (8 Hrs old)
Sterile disc (Whatman No.1 filter paper)


Procedure:

1. Preparation of Muller Hinton agar (MHA) plate :
       38 gm of MHA medium suspend in 1 liter of distilled water. pH is neutral.

       Heat (boil) and agitate frequently to dissolve.

       Autoclave the medium 121° c for 15 min.

       Pour into sterile petri dishes. 

2. Swabbing bacterial suspension (0.5 McFarland std. or equal to 1-2x10⁸   CFU/mL) on agar pales  

    by sterile cotton swabs.

3.Stock solution of plant extracts/ other sources to prepare at required concentration. (Eg.    concentration of solution 1mg/mL).

4. Soak the sterile discs in 10-50 µL (desired volume) of plant extracts.

5. Place the discs on agar medium.

6. Keep positive control (standard antibiotic disc) and negative control (eg.DMSO) on the plate.

7. Incubate the plate at 37° c for 24 Hrs.

8. After the incubation the diameter of zone of inhibition (mm) to be measured. 

Ref:
NCCLS