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The agar well diffusion assay

DN Dereje Nigussie
GD Gail Davey
BL Belete Adefris Legesse
AF Abebaw Fekadu
EM Eyasu Makonnen
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The Kirby-Bauer technique was used to determine the antibacterial activity of the extracts [11]. A total of 11 bacteria strains were used for this test. Mueller Hinton agar (pH 7.2 & 4 mm depth) plates were inoculated with test organisms (prepared in a sterile saline tube) by streaking the loop in a back-and-forth motion to ensure an even distribution of inoculum. MHA with 5% sheep blood was used for Streptococcus pyogenes. A circular 6 mm diameter well was punched aseptically with a sterile cork borer. Then, a volume of 100 μL methanol extracts of A. aspera, L. inermis and A. indica leaves (at concentrations of 100 mg/mL, 200 mg/mL, and 400 mg/mL) were dispensed into the wells. Similarly, 5% Di-methyl-sulfoxide (DMSO) was dispensed into the control well, and reference antibiotic discs were placed on the surface of the plate and incubated for 24 h at 37 °C. For Streptococcus pyogenes, a carbon dioxide incubator was used for incubation. Each experiment was done three times.

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