full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 993-997 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)994heating (Electrolux EM30EC90SS) at 720 W.Total heating time was kept 90, 70, and 180 secondfor methanol, ethanol, and chloroform respectively,with intermittent cooling. This was followed bycentrifugation (at 10,000 rpm for 15 min.), andfiltration with Whatman paper # 1 (WhatmanInternational Ltd., Maidstone, England). Solventwas evaporated from the filtered extract and thenthe dried extracts were reconstituted in: (i) theirrespective solvents for disc diffusion assay, and(ii) dimethyl sulfoxide (DMSO) for broth dilutionassay. Reconstituted extracts were stored underrefrigeration for further use. Extraction efficiencywas calculated as percentage weight of thestarting dried plant material. Extraction efficiencyranged from 2.8-13.6 %, with highest (13.6%)being in case of methanol extract of T. dioicaseeds.Bacterial strains: Staphylococcus aureusMTCC 737, Streptococcus pyogenes MTCC442, Staphylococcus epidermidis MTCC 435,Escherichia coli MTCC 723, Aeromonashydrophila MTCC 1739, Salmonella paratyphiA, Shigella flexneri MTCC 1457, Vibriocholerae MTCC 3906, and Pseudomonasoleovorans MTCC 617. S. paratyphi A wasprocured from Department of Microbiology,Gujarat University, Ahmedabad, while the restwere obtained from Microbial Type CultureCollection (MTCC), Chandigarh.Disc diffusion assay (DDA) : This wasperformed by Kirby-Bauer method as perNCCLS guidelines (5). 500 µL of inoculum(adjusted to 0.5 McFarland standard) was spreadon surface of Muller-Hinton agar medium(HiMedia, Mumbai, India). Sterile discs (6 mmdiameter) made of Whatman paper # 1 weredipped into the test extract and were put onto theagar surface after complete drying. Discs dippedinto pure solvents (separate disc for each solvent)after drying were applied as negative control.Commercially available discs of eitherstreptomycin or Ofloxacin (HiMedia) served aspositive control. Plates were then incubated at35ºC for 24 h. After incubation plates wereobserved for zones of inhibition, and their diameterwere measured. Studies were performed intriplicates.MIC determination : MIC (minimum inhibitoryconcentration) determination was carried outusing microbroth dilution method as per NCCLSguidelines (5). Assay was performed in a 96-wellmicrotitre plate. Total volume of the assay systemin each well was kept 200 µL. Cation-adjustedMuller-Hinton broth (HiMedia) was used asgrowth medium. Inoculum density of the testorganisms was adjusted to that of 0.5 McFarlandstandard. Broth was dispensed into wells ofmicrotitre plate followed by addition of test extractand inoculum. Extracts (reconstituted in DMSO)were serially diluted into each of the wells. ADMSO control was included in all assays (6).Gentamicin (HiMedia) served as positive control.Plates were incubated at 35ºC for 16-20 h, beforebeing read at 655 nm in a plate reader (BIORAD680). MIC was recorded as the lowestconcentration at which no growth was observed.All MICs were determined on three independentoccasions. Concentration at which growth wasinhibited by 50% was recorded as IC 50value.After reading the plates for MIC, subculturingwas made on nutrient agar plate from the wellsshowing no growth, so as to determine whetherthe extract is bactericidal or bacteriostatic. Growthon the plate indicated bacteriostatic action,absence of growth indicates bactericidal action.Total activity: Total activity (mL/g) wascalculated as (7): Amount extracted from 1 g (mg)/ MIC (mg/mL).Activity index: Activity index was calculated as(8)- zone of inhibition by extract / zone ofinhibition by antimicrobial agent used as positivecontrolVijay Kothari