IJPRD, 2011; Vol 4(06): August-2012 ( ) International Standard Serial Number

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1 IJPRD, 2011; Vol 4(06): August2012 ( ) International Standard Serial Number ISOLATION OF ACTINOMYCETES FROM DIFFERENT SOIL SAMPLES COLLECTED FROM MEDICINAL PLANTS AREA Rashmi Tripathi* 2, M.B.Patil 3, Umesh Kumar 1, Ashish Tripathi 1, D.A.Jain 2 1 College of Pharmacy, Agra, Uttar Pradesh 2 Institute of Pharmaceutical Sciences and Research Center, Bhagwant University, Ajmer, Rajasthan, India 3 G.S. Moze college College of Pharmacy Pune, India ABSTRACT The greatest variety of antibiotics is produced by Actinomycetes among all microbes. More than 50% of the known natural antibiotics produced by Actinomycetes. Two thirds of the present day antibiotics are of Actinomycetes origin like Aminoglycosides, Chloramphenicol, Macrolides, Tetracyclines, Nucleosides, Peptides and Polyenes.Till 1974 antibiotics of Actinomycetes origin were almost exclusively confirmed to Streptomyces. Actinomycetes are a diverse group of heterotrophic prokaryotes forming hyphae at some stage of their growth hence referred to as filamentous prokaryotes. They have different morphological, cultural, biochemical and physiological characters. This group is a potential producer of many enzymes, enzyme inhibitors, growth promoting substances and antibiotics. Different soil sample were collected into sterile plastic bags and were kept in aseptic condition at different time interval near the different medicinal plants of different regions of India. Soil samples were serially diluted upto 10 6 and 1 ml from each dilution were plated on different isolation media like starch Casein agar, Albumin media and YMA media. consisting of antifungal agent Nystatin 50 µg/ ml, by pour plate technique. The plates will be incubated at different temperature upto 710 days. There were 18 Actinomycetes isolated from Rose soil, Ashoka soil etc. The potent actinomycetes were characterized by morphological and biochemical methods. Correspondence Author Rashmi Tripathi Institute of Pharmaceutical Sciences and Research Center, Bhagwant University, Ajmer, Rajasthan, India umeshnandi211@gmail.com KEYWORDS : Actinomycetes, Soil, Starch Casein Agar, Albumin media and YMA media etc. 195

2 INTRODUCTION 1, 14 ACTINOMYCETES Actinomycetes are a diverse group of heterotrophic prokaryotes forming hyphae at some stage of their growth hence referred to as filamentous prokaryotes. They have different morphological, cultural, biochemical and physiological characters. This group is a potential producer of many enzymes, enzyme inhibitors, growth promoting substances and antibiotics. Actinomycetes are gram ve bacteria belonging to the order actinomycetales characterized by the formation of substances and aerial mycelium on solid media, presence of spores and a high GC content of DNA (6070 mol%). The composition of the growth media can be profound by affect the growth and stability of substrate and aerial mycelium The greatest variety of antibiotics is produced by Actinomycetes among all microbes. More than 50% of the known natural antibiotics produced by Actinomycetes. Two thirds of the present day antibiotics are of Actinomycetes origin like Aminoglycosides, Chloramphenicol, Macrolides, Tetracyclines, Nucleosides, Peptides and Polyenes.Till 1974 antibiotics of Actinomycetes origin were almost exclusively confirmed to Streptomyces. Nowadays efforts are being made to explore rare actinomycetes like Actinomadura Actinoplanes Actinosynnema Dactylosporangium Kibdilosporangium etc. IMPORTANT MICROBES PRODUCING ANTIBIOTICS 4 S. No. NAME OF NAME OF ANTIBIOTICS MICROORGANISM 1 P. notatum Penicillin 2 P. griseofulvum Griseofulvin 3 P. chrysogenum Penicillin 4 S. griseus Streptomycin 5 S. venezuelae Chloramphenicol 6 S. aureofaciens Chlortetracycline 7 S. virdofaciens Aureomycin 8 S. rimosus Oxytetracycline 9 S. texas Tetracycline 10 S. aureofaciens Dimethylchlortetracycline 11 S. erythricas Erythromycin 12 S. halstedii Carbamycin 13 S. ambofaciens Ravomycin 14 S. noursei Nystatin 15 S. griseus Cycloheximide MATERIALS AND METHODS: Collection of sample: There were 15 soil sample were collected from different regions of India and stored into sterile plastic bags and were kept in aseptic condition. Isolation of actinomycetes from soil samples: 5, 7 Soil samples were serially diluted upto 10 6 and 1 ml from each dilution were plated on different 196

3 isolation media like starch Casein agar, Albumin media YMA media etc. consisting of antifungal agent Nystatin 50 µg/ ml, by pour plate technique. The plates will be incubated at different temperature upto 710 days. IDENTIFICATION OF DIFFERENT ACTINOMYCETES Gram s staining The microbes smear were taken on glass slide. The smear were air dried. Smear were covered with crystal violet for 30 seconds. Covered each smear with Gram s Iodine solution for 60 seconds. Washed off Iodine solution with 95% ethyl alcohol, ethyl alcohol was added drop by drop until no more colour flows from the smear. The slides were washed with distilled water and drain. Safranin was applied to smears for 30 seconds (counter staining). The slides were washed with distilled water and blot dried with absorbent paper. Let the stained slides air dry. The slides were examined under microscope. Biochemical Test Certain biochemical test were performed for identification of different strains producing antibacterial compound. Melanoid Formation Test 0.5 gm Ltyrosine was Suspended in 10 ml distilled water in culture tube mixed thoroughly by vortexing and autoclave at C (15 lbs) for 15 min. 100ml base was combined (Beef extract3 gm, Peptone5 gm, Agar15 gm, distilled water1000 ml), mixed thoroughly by gentle rotation of bottle 2 or 3 times. Aseptically 35 ml of media was dispensed into tubes with frequent mixing. Tubes were cooled rapidly to prevent separation of tyrosine. The isolated actinomycetes were inoculated and kept for incubation at 37 0 C for 4 days. Test for Nitrate Reduction The composition of Organic nitrate broth media weighed and dissolved in required quantity of distilled water and sterilized at C (15 lbs) for 15 min by using autoclave. After sterilization the media were poured into Test tubes under sterile condition (laminar air flow) and allow to cool. The isolated actinomycetes were inoculated and kept for incubation at 37 0 C for 4 days. After incubation, 1 ml of broth was taken and added 2 drops of alphanaphthalene, 23 drops of H 2 SO 4 and observed for any change in colour. o Test for Acid Production The composition of Glucose nutrient broth media were weighed and dissolved in required quantity of distilled water and sterilized at C (15 lbs) for 15 min by using autoclave. After sterilization the media were poured into Test tubes under sterile condition (laminar air flow) and allow to cool. The isolated actinomycetes were inoculated and kept for incubation at 37 0 C for 4 days. The broth was checked daily for any change in colour. Hydrogen Sulphide Production Test 197

4 The composition of SIM media were weighed and dissolved in required quantity of distilled water and sterilized at C (15 lbs) for 15 min by using autoclave. After sterilization the media were poured into Test tubes under sterile condition (laminar air flow) and allow to cool. Inoculated the isolated actinomycetes into its appropriately labeled tube by means of stab inoculation. Incubated the inoculated tubes at 35 0 C.for about 48 hrs. Examined the tubes for the presence or absence of black coloration along the line of stab inoculation. RESULTS AND DISCUSSION The actinomycetes isolated from different soil on different media are shown as actinomycetes were isolated from 15 soil samples. The actinomycetes isolated from plants were shown in photograph 15. Photograph 1 : Showing Actinomycetes isolated from Atropa on Albumin Media S.No. Name of the plants soil used Nutritional Media 1 Atropa soil Albumin Four 2 Cardamom soil Albumin Three 3 Ashoka soil Albumin YMA Two Two 4 Citrus Soil Albumin Two No. of actinomycetes isolated Photograph 2 : Showing Actinomycetes isolated from Ashoka on YMA Media 5 Syzineum YMA Three Soil 6 Rose Soil Albumin Two 7 Aloe vera YMA Three Soil Table No.1: Showing total number of actinomycetes isolated from different soil samples. Out of 15 plants soil only seven having Actinomycetes and So. there were 18 Photograph 3: Showing Actinomycetes isolated from Ashoka on Albumin Media 198

5 Photograph 4: Showing Actinomycetes isolated from Syzineum on YMA Media Photograph 5: Showing Actinomycetes isolated from Rose on Albumin Media Media AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 ISP2 ISP4 ISP5 ISP6 ISP7 Table No.2: Showing actinomycetes growth on different ISP media isolated from different soil samples. Media AA10 AA11 AA12 AA13 AA14 AA15 AA16 AA17 AA18 ISP2 ISP4 ISP5 ISP6 ISP7 Table No.3: Showing actinomycetes growth on different ISP media isolated from different soil samples. Actinomycetes strain Reaction AA1 AA2 ve ve 199

6 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10 AA11 AA12 AA13 AA14 AA15 AA16 AA17 AA18 ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Table No.4. Showing the Melanoid formation test. Actinomycetes strain Reaction AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10 AA11 AA12 AA13 AA14 AA15 AA16 AA17 AA18 ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Table No. 5. Showing Nitrate reduction test. 200

7 Actinomycetes strain AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10 AA11 AA12 AA13 AA14 AA15 AA16 AA17 AA18 Reaction ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Table No.6. Showing Acid production test. Actinomycetes strain Reaction AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10 AA11 AA12 AA13 AA14 AA15 AA16 AA17 AA18 ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve ve Table No. 7.Showing Hydrogen sulphide test. 201

8 Our study prove that actinomycetes may be isolated from different soil samples surrounding the medicinal plants and which may be further study for the production of different useful compounds like Antibiotics, Enzymes and Xenobiotics etc. REFERENCES 1. Ozgur C, Gulten O and Aysel U. Isolation of soil Streptomyces as source antibiotics active against antibioticresistant bacteria. Eur Asian Journal of BioSciences.2008; 2: Chi NS, Ji H C, and Keun S B. An Improved selective isolation of rare actinomycetes from forest soil. The Journal of Microbiology. 2001: Mustafa O. Comparison of Streptomyces diversity between agricultural and nonagricultural soils by using various culture media. Scientific Research and Essay. 2009; 4: Sivakumar K. Centre of advanced study in marine biology. Annamalai University : Lo CW, Lai NS, Cheah HY, Wong NKI. and Ho CC. Actinomycetes isolated from soil samples from the crocker range sabah. ASEAN Review of biodiversity and environmental conservation; 2002: Singh SL and Bora T C. Actinomycetes of Loktak habitat.isolation and screening of antimicrobial activity. Biotechnology.2006; 5: Deepika L and Krishnan T. A report on antidermatophytic activity of actinomycetes. IIJB. 2009: Manjula C, Rajguru P and Muthuselvam M. Screening for antibiotic sensitivity of free and immobilized actinomycetes isolated from India. Advances in Biological Research. 2009; 3: Srivibool R, Kurakami K, Sukchotiratana M and Tokuyama S. Coastal soil actinomycetes:thermotolerant strains producing n acylamino acid racemase. ScienceAsia. 30 ;2004: Sultan MZ, Khatune NA, Sathi ZS, Md. Shah and Bhuiyan A. Biotechnology. 2002; 1: Jongrungruangchok S, Tanasupawat S, Kitakoop P, Bavovada R, Kobayashi H, and Kudo T. Identification of streptomyces and kitasatospora strains from thai soils with geldanamycin production strain. Actinomycetologica. 2006: 20: Igarashi Y, Miura S, Fujita T and Furumai T. Pterocidin :A Cytotoxic Compound from the Endophytic Streptomyces hygroscopicus. Journal of Antibiotics. 2006; 59: Wenhan L, Liya Li, Hongzheng Fu, Isabel S, Xueshi H and Susanne G. New Cyclopentenone Derivatives from an Endophytic Streptomyces sp. Isolated from the Mangrove Plant Aegiceras comiculatum. J. Antibiot. 2005; 58: Gary S and Bryn D. Bioprospecting for Microbial Endophytes and Their Natural Products. Microbiology and molecular biology reviews. 2003;4: Oskay M, Tamer A. U and Azeri C. Antibacterial activity of some actinomycetes isolated from farming soils of Turkey. African Journal of Biotechnology. 2004; 3: ***** 202