In this investigation, screening of different solvent extracts of Acacia nilotica for their antibacterial activity were carried out by disc diffusion method (Bauer et al., 1966). After initial screening minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined.
Collection of plant materials and preparation of solvent extracts: The fresh leaves of Acacia nilotica were collected from the campus of Khulna University. Powdered material was extracted successively with two different solvents using the concept of the nature of solubility and distribution of the active ingredients. For this, methanol and hexane were used as polar and non-polar solvent respectively. Powder was mixed with methanol and preserved for several days in an airtight flask. The liquid portion of the mixture was taken into a separation funnel and the debris was discarded. In separation funnel, appropriate amount of hexane was given and occasionally shaken several times. The hexane portion was then separated and collected in beaker and this procedure was repeated three times. Liquid portion in the beaker was air-dried. In the same way methanol extract was obtained using methanol portion of the separation flux.
Screening of Acacia nilotica leaf extracts for antibacterial activity against pathogenic bacteria: The antibacterial activity of Acacia nilotica leaf extracts was done by the disc diffusion method (Bauer et al., 1966). Hospital isolates of ten pathogenic bacteria were collected from International Center for Diarrhoeal Disease Research’ Bangladesh (ICDDR’B) for the test.
Preparation of the discs: Paper discs were placed in a sterile blank petridish. The discs were impregnated separately with the solution of plant extracts so that each disc contain 1000 µg of the test material and left for a period of time in an aseptic condition for the complete evaporation of the solvents. Control discs were also prepared in order to determine whether remaining solvent (if any) in disc shows any antimicrobial activity. For the preparation of control disc equal volume of solvent without test material was used to soak the paper disc. Kanamycin was used as standard antibiotic disc (Oxoid Ltd).
Procedure of disc diffusion method for screening antibacterial activity: From overnight culture plate, small portion of a fresh colony was transferred to test tube containing Muller-Hinton broth and incubated at 37 °C until the growth reached the log phase (~5×106 cfu ml-1). After optimum growth, plates were seeded properly by pouring the culture broth with a pasteur pipette to make a bacterial lawn. The excess culture-broth were discarded from the plates with a pasteur pipette and allowed to dry for 5 minutes. Discs impregnated with different solvent extracts were placed at a proportionate distance from each other using a sterile needle. The plates were incubated overnight at 37 °C and zone of inhibition (if any) was measured in mm.
In vitro determination of minimum inhibitory concentration (MIC) of the Acacia nilotica leaf extracts: The minimum inhibitory concentration (MIC) of methanol extract was determined by the broth dilution test tube method (Blair et al., 1997). In brief, an increasing concentration of solvent extracts (200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1200 µg ml-1) were added to different test tube, each containing 3 ml of tryptone soy broth. Then 10 µl inoculum from young liquid culture (~5×106 cfu ml-1) was added. Additional test tubes were also included as control to check the sterility of the media, inhibitory effect of the solvent itself and viability of inoculum. All the tubes were incubated overnight at 37 °C and checked for growth. Tubes with lowest concentrations of extracts without any turbidity were considered MIC of the respective solvent extracts. However, in case of colored extract, subculture was made from the tubes to determine the MIC.
In vitro determination of Minimum Bactericidal Concentration (MBC) of methanol and hexane extracts: The minimum bactericidal concentration (MBC) of solvent extracts were determined by measuring the viability loss of bacterial culture with increasing concentrations of extract added. Test tubes containing higher concentrations of leaf extracts than MIC were plated out on to TSA plate to check the viability. Suspension from the tube containing lowest concentration of extracts producing no colonies after overnight incubation indicated MBC value.