Antimicrobial Activity - Agar Well Diffusion Method

Agar well diffusion method is to determine the antimicrobial activity (NCCLS). 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 cork borer

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.Wells to be made by using sterile cork borer (size of the well range between 6mm to 10mm    

     diameter).

4. Stock solution of plant extracts/ other sources to prepare at required concentration. (eg.  

    concentration of solution 1mg/mL) and  add into the well (volume 50 -100µL range).

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

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

7. Three replicates to be performed against each organism.

Ref:
NCCLS