Determination of antibacterial activity
The modified agar well-diffusion method6 was conducted to evaluate the antibacterial activities of the extracts. A freshly grown culture was serially diluted, and 0.1 ml of prepared cells (1.5107 colony forming www.scienceasia.org 320 ScienceAsia 38 (2012) units per millilitre, CFU/ml) was aseptically spread onto the surface of Mueller Hinton agar and then
left to dry for 30 min. Wells (8 mm in diameter) were made in media using a sterilized stainless steel borer. Each well was filled with 30 μl of the crude extracts. The plates were left at room temperature for 30 min to allow diffusion of materials in media. The controls were prepared using residue obtained by concentrating each solvent under reduced pressure
(the same as done with the filtrates) and diluting with 10 ml DMSO. Plates were incubated at 37 °C for 18–24 h.
Inhibition zones in mm (including well diameter)around wells were measured. The antibacterial activity was expressed as the diameter of inhibition zones produced by the extracts against test bacteria. All tests were performed in triplicate. The results were expressed as meanstandard deviation. Statistical significance was calculated by ANOVA, followed by
Scheff´e’s test. The p values of < 0.05 were considered significant.