Plant materials and extracts preparation. Fresh and sound of Long pepper (Piper longum) and Water cress (Enhydra fluctuans) were collected from Meherpur, Magura, Faridpur and Kushtia districts. The leaves, stems and roots were taken as sample. The aqueous extraction of the water-soluble ingredients of the plant parts were carried out using the method as described by Asuzu. 15 g of each of the grounded plant part was extracted by successive soaking for 3 days using 35 ml of distilled water for each sample in separate container. The extracts were filtered using Whatman No 1 filter paper, after which the filtrates were concentrated by evaporation at low temperature of 30 °C using water bath. The concentrated extracts were stored in the refrigerator until required. Alternatively for aqueous extraction to be more effective, 10 g of air-dried powder was added to distilled water and boiled on slow heat for 2 hours. It was then filtered through 8 layers of muslin cloth and centrifuged at 5000g for 10 min and the supernatant was collected. This procedure was repeated twice. After 6 hours, the supernatant collected at an interval of every 2 hours, was pooled together. Filtrates obtained were then bulked and lyophilised to recover the residues as powder, or in a few cases, as sticky pastes, which were further dried in a desiccator. Aqueous extracts of the plants were employed in this study because the water was likely to extract the same principles as are extracted under inuse conditions. It was then autoclaved at 121 °C temperature and at 15 lbs pressure and stored at 40 °C. These processes of aqueous extraction were applied for all the four test species of plant.
For solvent extraction, 10 g of air-dried powder was taken in 100 ml of organic solvent (absolute ethanol or chloroform or acetone) in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours the supernatant was collected. Thus the ethanol, chloroform and acetone extracts were prepared separately from the air-dried powder of each of the four plant species. The three organic extract i.e. ethanol extract; acetone extract and chloroform extract were filtered with the aid of a Whatman No 1 filter paper. The extracts were then rotary evaporated to dryness at 40 0C. The concentrated extracts were stored in the refrigerator until required.
Microbial strains Five strains of bacteria were used for antibacterial evaluation against the extracts. Among the five strains, Staphylococcus aureus and Staphylococcus saprophyticus were gram positive and Escherichia coli, Salmonella typhi and Shigella dysenteriae were gram negative. The bacterial strains were maintained on agar slant at 4 oC in the microbiology laboratory of the Dept. of Applied Nutrition and Food Technology of Islamic University, Kushtia, where the antimicrobial tests were performed.
Culture media Muller-Hinton Agar and Nutrient Agar Media were used to assay antimicrobial activity and Minimum Inhibitory Concentration (MIC). Mueller-Hinton Agar and Nutrient Agar is considered to be the best for routine susceptibility testing of nonfastidious bacteria.
Preparation of Subculture The test organisms were transferred to the nutrient agar slants from the pure cultures with the help of an inoculation loop in aseptic condition using laminar air cabinet. For growth of the test organisms the inoculated slants were incubated at 37 0C for 18-24 hours in an incubator. These fresh cultures were used for sensitivity test within 2 to 3 days.
Preparation of Inoculums After incubation at 37 0C for 18-24 hours, well-isolated colonies were transferred into a sterile screw capped test tube containing sterile distilled water and vortex thoroughly. The bacterial suspension was then compared to 0.5 McFarland standards for turbidity standard. This results in a suspension containing 107 to 108 CFU/ml. The accuracy of the density of a prepared McFarland standard was checked by using a spectrophotometer with a 1-cm light path; for the 0.5 McFarland standard, the absorbance at a wavelength of 625 nm should be 0.08 to 0.1. If the bacterial suspension does not appear to be the same density as the McFarland 0.5, the turbidity can be reduced by adding sterile saline or increased by adding more bacterial growth.
Inoculation procedure Within 15 minutes after adjusting the turbidity of the inoculums suspension, a sterile cotton swab was dipped into the suspension. Pressing firmly against the inside wall of the tube just above the fluid level, the swab was rotated to remove excess liquid. Then the swab was streaked over the entire surface of the agar medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculums. Finally, the swab was streaked all around the edge of the agar surface.
Serial Dilution and Preparation of Sample Solution All the extracts including aqueous and organic extracts (ethanol extract, acetone extract and chloroform extract) of Long pepper (Piper longum) and Water cress (Enhydra fluctuans) were used for serial dilution. First 32 mg of extract were taken in a sterile screw capped test tube containing 2 ml solvent and mixed well immediately with vortex mixture to make the concentration of the solution 16 mg/ml. Then 1 ml of the sample solution having 16 mg/ml extract was transferred to the second sterile test tube and mixed well with another 1 ml solvent to make the volume 2 ml and concentration 8 mg /ml. This process of serial dilution was continued up to five test tubes; finally the concentration was found 16 mg/ml in the first test tube and 1 mg/ml in the last. Then 1024 µl of the solution from the last test tube containing 1 mg/ml or 1 µg/µl was transferred to another sterile test tube and 976 of solvent was added to the test tube to make the final volume 2 ml and concentration 512 µg/ml. The content of the test tube was mixed well with vortex mixture. This process of serial dilution was continued up to ten test tubes; finally the concentration was found 512 µg/ml in the first test tube and 1 µg/ml in the last test tube. The same procedure is followed for each of the extract extracted by different solvent.
Preparation of Discs Filter paper was punched with the punching machine to prepare discs approximately 6 mm in diameter, which was placed in a screw capped test tube and sterilized in an autoclave machine (15 minutes with 1210C temperature and 15 lbs inch-2 pressure). The sterilized paper disc was the soaked with sample solution of various concentration. The paper disc was then dried with a dryer. After drying the paper disc was labeled according to different concentration. Finally the labeled paper disc was taken into the sterile air tight vial and stored in refrigerator. It was then ready for use as sample disc. Punched, sterile paper disc (6 mm in diameter) was soaked with each solvent to prepare disc, which would be used as negative control. Standard antibiotic disc were purchased from Mast Diagnostic Centre, England. Following standard antibiotic disc were used as positive control.