@Katyayini Shirur....A clear zone around the well is indicative of antimicrobial activity of the compound/drug under investigation. If the zone is semi-transparent we consider it as bacteriostatic action however, a clear zone shows bactericidal action. In case of your experiment, it seems to me as bacteriostatic. Further it’s my sincere suggestion that whenever we are performing any such experiments we must have positive control (you can take any existing antimicrobial drug). As plant extracts have many chemical constituents with variable solubility/partitioning properties, sometimes in disc diffusion assay aberrant results are obtained. The activity solely depends on the diffusivity of the chemical constituent into the aqueous agar medium. As you are working with extracts, please ensure the chemical constituents and their contents for better results.

Best……

dear Katyayini Shirur,

as u have mentioned u did disc diffusion assay for the concentrated plant extract using Nutrient Agar medium. I will suggest you to perform experiment by Agar well diffusion method using Muller Hinton Agar. Dissolve the extract in 5%,8% or 20%DMSO (DMSO+distilled water). Add 20-40µl of the extract in the well and keep the plates  inside the refrigerator at 4°C for 6 hour to allow proper diffusion of extracts

into medium.Examine them for antibacterial activities after 24 hours of incubation at

37°C. Also take postive control (any antibiotic) and negative control (DMSO).

........

Dear Katyayini, I suggest to use broth dilution assay. As explained by Jay Gopal Meher, diffusibility of plant extracts from discs may change. For this reason, my suggestion is broth dilution asay by measuring OD600nm, you can follow bacterial growth inhibition in the presence of different concentrations of plant extracts. As explained by Aarti Bains, agar dilution assay is also applicable for plant extracts.

Best regards.

Hi Katyayani,

Agar well diffusion method using MHA plates should be used instead of nutrient agar plates for qualitative detection of antimicrobial property of plant extracts. If your extract has antimicrobial activity(qualitatively), you can quantify the activity using broth/agar dilution methods.Also, do use a DMSO (solvent) control along with the extract for testing the antimicrobial activity. 

Thank you @Jay Gopal Meher, @Aarti Bains, @Mert Sudagidan and @Chaitanya Tellapragada.

I will consider your suggestions and do the assay..