full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1043-1053 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1044against bacterial and fungal pathogens and theselected isolates were characterized on the basisof their morphological, physiological andbiochemical characteristics.Materials and MethodsCollection of soil samples: Soil samples werecollected from various locations i.e. rhizosphereof plants, agricultural soil, preserved areas andforest soils of Durg District of Chhattisgarh. Thesamples were taken from up to 20 cm depth, afterremoving approximately 3 cm of the soil surface.The samples were placed in sterile polyethylenebags, closed tightly and stored in a refrigerator at4°C until further analysis.Isolation of Actinomycetes: Soil samples wereincubated at 37°C for 4 days then suspended insterile ringer solution. Test tubes containing a 10 -2to 10 -6 dilution of samples were placed in a waterbath (SONAR, INDIA) at 45°C for 16 h toseparate spores from vegetative cells.Streptomyces were isolated by spread platetechnique by serial dilution of soil samples onstarch casein nitrate agar plates containingcycloheximide and nystatin, each at concentrationof 50 ìg/ml of medium to inhibit the fungal growth(11, 25). The plates were incubated at 28°C untilthe sporulation of Streptomyces colonies occurred.Pure cultures were obtained from selectedcolonies by repeated sub culturing on starch caseinnitrate agar slants.In-vitro screening of isolates for antagonism:Primary screening of isolates were done by usingof starch casein nitrate agar plates and inoculatedwith Streptomyces isolate by a single streak ofinoculum in the center of the Petri dish. After 4days of incubation at 28°C the plates were seededwith test organisms by a single streak at a 90°angle to the Streptomyces isolates. The microbialinteractions were analyzed by determination ofthe size of the inhibition zone (12). Isolatesshowing activity against test organisms weregrown in 250 ml flasks containing 50 ml of starchcasein nitrate broth medium. Seven-day-oldculture grown on starch casein nitrate agar mediawas used to inoculate the flasks. These flaskswere incubated in a rotary shaker at 100 rpm in28°C for seven days. The resulting culture broth(approximately 50 ml) was separated from themycelium by centrifugation at 8000rpm for 15 min.The supernatant obtained was used forextracellular antimicrobial activity by agar welldiffusion method (13). Appropriate agar plateswere seeded with the test organisms and wellswere made by using a sterile cork borer (6mm).One hundred micro liter of supernatant of eachisolate was loaded in each well. Plates were keptin refrigerator for about 1h to allow the diffusionof produced antimicrobial metabolites. Thediameters of inhibition were measured after 24 hof incubation at 37°C for bacteria, for 48 h ofincubation at 28°C for yeasts and filamentousfungi. Each experiment was repeated three timesand means value of inhibition zones wascalculated.Test organisms used: Test organisms used forscreening of antimicrobial activity of isolates, wereobtained from Microbial Type Culture Collectionand gene bank (MTCC), IMTECH Chandigarh.they are Bacillus subtilis (MTCC 1789), Bacillusmegaterium (MTCC 1684), Bacillus cereus(MTCC 1305), Staphylococcus aureus (MTCC96), Staphylococcus epidermis (MTCC 435),Salmonella typhi (MTCC 531), Proteus vulgaris(MTCC 1771), Klebsiella pneumoniae (MTCC2405), Escherichia coli (MTCC 1667), Candidaalbicans (MTCC 1637), Aspergillus niger(MTCC 872), Alterneria alternate (MTCC1779), Penicillium citrinum (MTCC 1751), fromJawaharlal Nehru Medical College (JNMC),Raipur, Staphylococcus aureus (JNMC),Singh and Rai