Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1043-1053 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Isolation <strong>and</strong> Characterization <strong>of</strong> Streptomyces sp. from DurgDistrict <strong>of</strong> Chhattisgarh for Antimicrobial activityN. Singh* <strong>and</strong> V. RaiMicrobiology <strong>and</strong> Biochemistry Lab, School <strong>of</strong> Studies in Life SciencesPt. Ravishankar Shukla University, Raipur-492010, Chhattisgarh, India*For Correspondence - nevi0007@gmail.com1043Abstract:The present work was carried out in orderto establish a suitable protocol for screeningprogram for antimicrobial producingStreptomyces sp. from soil sample <strong>of</strong> Durg District<strong>of</strong> Chhattisgarh, INDIA. Fifty sevenStreptomyces isolates with distinct characteristicswere isolated <strong>and</strong> purified by serial dilution methodon starch casein nitrate agar medium. Theseisolates were screened for their antimicrobialactivity against twenty one test organismsobtained from the Microbial Type CultureCollection <strong>and</strong> Gene Bank (MTCC), IMTECH,Ch<strong>and</strong>igarh. Morphological, physiological <strong>and</strong>biochemical characteristics indicated that theseisolates belong to the genus Streptomyces <strong>and</strong>characterized as Streptomyces rimosus,Streptomyces chromogenus, Streptomycesrouchi-3, Streptomyces antibioticus-3,Streptomyces filipinensis, Streptomycesatroolivaceus, Streptomyces exfloliatus-2,Streptomyces halstedii, Streptomyces cyaneus,Streptomyces violaceus, Streptomycesoliovaceoviridis, Streptomyces lydicus,Streptomyces phaeochromogenes,Streptomyces chrom<strong>of</strong>uscus . Among fifty sevenisolates, nineteen exhibited antimicrobial activityagainst test pathogens, <strong>and</strong> out <strong>of</strong> these nineteenisolates, 11 isolates exhibited both antibacterial<strong>and</strong> antifungal activity while 8 exhibited onlyantibacterial activity.Key words: Streptomyces sp., Antimicrobialactivity, Screening, CharacterizationIntroductionThe genus Streptomyces was described forthe first time by Waksman <strong>and</strong> Henrici in the year1943 (1). Streptomyces are filamentous, aerobicspore formers <strong>and</strong> omnipresent (1, 2). Presence<strong>of</strong> L, L isomer <strong>of</strong> 2, 6-Diaminopimelic acid (L,L-DAP) <strong>and</strong> absence <strong>of</strong> any diagnostic sugar in thecell wall is a salient feature <strong>of</strong> the genusStreptomyces (3). Streptomycetes proved to bea rich source <strong>of</strong> novel bioactive, commerciallysignificant compounds (4, 5, 6). Though genusStreptomyces is being studied from last manydecade still there is very little documentedinformation <strong>of</strong> the occurrence <strong>of</strong> Streptomycessp. from the different soil habit (5,7,8,9).Streptomyces is the largest antibiotic-producing(almost 75% <strong>of</strong> commercially <strong>and</strong> medicallyuseful antibiotics) genus in the microbial worlddiscovered so far (2). The number <strong>of</strong> antimicrobialcompounds reported from the species <strong>of</strong> this genusper year has increased almost exponentially forabout two decades. Reports show that this group<strong>of</strong> microorganisms still remains an importantsource <strong>of</strong> antibiotics (2), because they are readilybiodegradable, specific <strong>and</strong> generally have lowtoxicity (10). In the present study, Streptomycesisolated from soil <strong>of</strong> Durg District <strong>of</strong> Chhattisgarhwere screened for their antimicrobial activityIsolation <strong>and</strong> Characterization <strong>of</strong> Streptomyces sp.
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1043-1053 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1044against bacterial <strong>and</strong> fungal pathogens <strong>and</strong> theselected isolates were characterized on the basis<strong>of</strong> their morphological, physiological <strong>and</strong>biochemical characteristics.Materials <strong>and</strong> MethodsCollection <strong>of</strong> soil samples: Soil samples werecollected from various locations i.e. rhizosphere<strong>of</strong> plants, agricultural soil, preserved areas <strong>and</strong>forest soils <strong>of</strong> Durg District <strong>of</strong> Chhattisgarh. Thesamples were taken from up to 20 cm depth, afterremoving approximately 3 cm <strong>of</strong> the soil surface.The samples were placed in sterile polyethylenebags, closed tightly <strong>and</strong> stored in a refrigerator at4°C until further analysis.Isolation <strong>of</strong> Actinomycetes: Soil samples wereincubated at 37°C for 4 days then suspended insterile ringer solution. Test tubes containing a 10 -2to 10 -6 dilution <strong>of</strong> 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 <strong>of</strong> soil samples onstarch casein nitrate agar plates containingcycloheximide <strong>and</strong> nystatin, each at concentration<strong>of</strong> 50 ìg/ml <strong>of</strong> medium to inhibit the fungal growth(11, 25). The plates were incubated at 28°C untilthe sporulation <strong>of</strong> Streptomyces colonies occurred.Pure cultures were obtained from selectedcolonies by repeated sub culturing on starch caseinnitrate agar slants.In-vitro screening <strong>of</strong> isolates for antagonism:Primary screening <strong>of</strong> isolates were done by using<strong>of</strong> starch casein nitrate agar plates <strong>and</strong> inoculatedwith Streptomyces isolate by a single streak <strong>of</strong>inoculum in the center <strong>of</strong> the Petri dish. After 4days <strong>of</strong> 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 <strong>of</strong>the size <strong>of</strong> the inhibition zone (12). Isolatesshowing activity against test organisms weregrown in 250 ml flasks containing 50 ml <strong>of</strong> 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 <strong>and</strong> wellswere made by using a sterile cork borer (6mm).One hundred micro liter <strong>of</strong> supernatant <strong>of</strong> eachisolate was loaded in each well. Plates were keptin refrigerator for about 1h to allow the diffusion<strong>of</strong> produced antimicrobial metabolites. Thediameters <strong>of</strong> inhibition were measured after 24 h<strong>of</strong> incubation at 37°C for bacteria, for 48 h <strong>of</strong>incubation at 28°C for yeasts <strong>and</strong> filamentousfungi. Each experiment was repeated three times<strong>and</strong> means value <strong>of</strong> inhibition zones wascalculated.Test organisms used: Test organisms used forscreening <strong>of</strong> antimicrobial activity <strong>of</strong> isolates, wereobtained from Microbial Type Culture Collection<strong>and</strong> gene bank (MTCC), IMTECH Ch<strong>and</strong>igarh.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), C<strong>and</strong>idaalbicans (MTCC 1637), Aspergillus niger(MTCC 872), Alterneria alternate (MTCC1779), Penicillium citrinum (MTCC 1751), fromJawaharlal Nehru Medical College (JNMC),Raipur, Staphylococcus aureus (JNMC),Singh <strong>and</strong> Rai