Plant Material The leaves of E. hirta were collected from Kushtia district of Bangladesh during the month of July, 2011. This plant was then botanically identified by taxonomists and the name of the plant, time, place, and date of collection were recorded. The leaves were initially rinsed with distilled water and dried on a paper towel in laboratory under shade and used for the present study.
Preparation of the Extract Collected leaves were cleaned with deionized water and dried in shade and pulverized into fine powdered substances by a grinding machine. Each 30 g of powder was transferred into two separate 100 mL conical flasks. Then each 40 mL of methanol and ethanol (MerckLimited, India) were added in the flasks respectively, closed by foil paper and placed on a shaker at 37 °C temperature for 24 h. The crude extracts were then filtered by passing the extracts through Whatman No. 1 filter paper (UK) and then concentrated under vacuum at 40 °C by using a rotary evaporator. The standard extract obtained was then stored in a refrigerator at 4 °C for further use.
Test Bacteria Pure culture of three Gram-negative, i.e., Escherichia coli, Pseudomonas sp, Klebsiella pneumoniae and two Gram-positive, i.e., Bacillus subtilis, Sarcina lutea and Xanthomonas campestris bacterial isolates were obtained from the microbial type culture collection (MTCC) of Microbiology Laboratory of the Biotechnology and Genetic Engineering Department, Islamic University, Kushtia-7003, Bangladesh. The test bacteria were cultured on nutrient agar (Hi-Media, India) at 37 C for 24 h.
Bacterial Culture Media For the cultivation and maintenance of different bacterial culture and for the identification and microbial sensitivity, nutrient agar (Hi-Media, India) was used. Lactose broth (LB) media was used for culturing of the bacteria. Lactose broth is also used for the detection of coliform organisms in water, dairy products, and other materials.
Inoculum Preparation The OD (optical density) was measured with a spectrophotometer at a wavelength of 530 nm and bacterial population was confirmed to be within 107 mL −1 to 108 mL−1 and then plated out as inoculums.
Antibacterial Activity The antibacterial activity of the test samples was tested by disc diffusion method. The filter paper discs of 6 mm diameter were prepared using Whatman No. 1 filter paper (UK), soaked in extract and incubated for 17 h at room temperature for the purpose. The discs dipped in respective solvent were used as negative controls. The antibacterial agent cloxacillin was used as standard. The petridishes were sterilized in hot air oven and nutrient agar medium was sterilized by autoclaving. This media was poured in the sterile petri-dishes and 1 mL of bacterial culture was added. The impregnated discs were aseptically placed on the solidified agar media. The plain discs and standard were also placed on the solidified agar media. After 24 h of incubation at 37 °C temperature the culture plates were examined and the diameters of the inhibition zones were measured in mm unit. Minimum inhibitory concentration (MIC) was determined in the present study following the serial dilution technique.
Statistical Evaluation The antibacterial activity was determined by measuring the diameter of the zone of inhibition that is the mean of triplicates ± SD (standard deviation).