The young leaf and stem (twig) of L. inermis L. and M. pudica L. were collected from Rajshahi University Flora, Bangladesh and identifi ed at the Department of Botany, Rajshahi University, Rajshahi-6205. These specimens are preserved in the herbarium, Department of Botany, Rajshahi University, Rajshahi-6205, Bangladesh (Lawsonia inermis L.- Voucher Specimen No. 36 and Mimosa pudica L.- Voucher Specimen No. 52).
Following Gram-positive Staphylococcus aureus (BMLRU1002), Bacillus cereus (BMLRU1004), Staphylococcus haemolytica (BMLRU1006), Bacillus subtilis (BMLRU1008), Bacillus megaterium (BMLRU1010), Sarcina lutea (BMLRU1012) and Gram-negative Escherichia coli-B (BMLRU1001), Klebsiella sp. (BMLRU1003), Klebsiella pneumoniae (BMLRU1005), Pseudomonas aeruginosa (BMLRU1007), Salmonella typhi (BMLRU1009), Shigella dysenteriae (BMLRU1011), Shigella shinga (BMLRU1013), Shigella sonnei (BMLRU1015) and Pseudomonas sp. (BMLRU1017) species were tested. The strains were collected from the International Center for Diarrhoeal Disease Research of Bangladesh (ICDDRB), Mahakhali, Dhaka, Bangladesh.
Cleaned fresh twig samples of Lawsonia and Mimosa were cut into small pieces, oven dried at 40°. Well pulverized 5 g powder of the plant sample was continuously shaken with 20 ml each of ethanol, petroleum ether and chloroform on a water bath shaker for 10 h. The extracts were filtered through the Whatman No.1 filter paper, and the solvents were evaporated using a rotary evaporator to obtain brownish dark sticky residues. The evaporated extracts were dissolved in the respective solvents (10 mg/ml).
Bacterial strains were grown on Luria-Bertani (LB) medium which was prepared using the following compositions: 10 g of yeast extract supplemented with 10 g of bacto peptone and 5 g of NaCl dissolved in 1 liter of water[2]. The pH was adjusted at 7.0 before solidifying with bacto agar (20 g/l). The medium was autoclaved at 15 lb/inch2 in pressures at the temperature of 121° for 20 min to ensure sterilization.
Disc diffusion method was followed to test of the antimicrobial activity of L. inermis and M. pudica against the chosen 15 bacterial strains. Discs of 6 mm in diameter were soaked with 10 μl of each extract (10 mg/ml) and air dried under aseptic condition inside the laminar flow and placed on seeded LB agar plates and incubated at 37° for 24 h. A 100 μl of bacterial suspension (108 cfu/ml) was used for spreading on LB agar plates. Only respective solvents (without extract) containing sterile blank discs were used as a negative control and tetracycline (30 μg/ml) was used as a positive control. After incubation, the antibacterial activity of L. inermis and M. pudica was determined by measuring zone of inhibition in millimeter scale against individual studied bacteria. Each assay was carried out in triplicate.
Two-folds serial dilution method was followed for MIC determination of studied plant extracts. Ten milligram of the semisolid extracts were dissolved in 2 ml of the respective solvent to get a concentration 5 mg/ml (5000 μg/ml) which was serially diluted to achieve 2500, 1250, 625, 312.5 and 156.25 μg/ ml, respectively. One hundred microlitters of each concentration of test samples were added into the test tubes containing 9 ml bacterial suspension (108 cfu/ml), separately. Control test tubes contained only test organisms with distilled water instead of plant extract. The test tubes were incubated at 37º for 24 h. The least concentration of the plant extracts with no visible growth was taken as the MIC.