I. List and composition of ISP media used in the present study for characterization of Streptomyces

Transcription

1 APPENDIX I I. List and composition of ISP media used in the present study for characterization of Streptomyces SL. MEDIA COMPONENT Bacto peptone Iron agar 36g Peptone yeast Iron agar (ISP 6) Bacto yeast extract 1g Tresner and Danga,1958 ph Yeast extract malt extract agar (ISP 2). Peridham et al., Glycerol asparagine agar (ISP5) Peridham and Lyons, 1961 Tyrosine agar (ISP 7) Shinobu, 1958 Inorganic salt starch agar (ISP 4). Kuster. 1995a Oat meal agar (ISP 3) Kuster. 1995a Bacto yeast extrac 4g,Bacto malt extract10g, Bacto dextrose4g L-asparagine 0 g, Glycerol 10.0g K2HPO4.0g,, ph 7-7.4, Agar 20g Glycerol 15g, L-tyrosine 0.5g L-asparagine 1g, K2HPO4 0.5g MgSO4.7H2O 0.5g,NaCl 0.5g FeSO4.7H2O 0.01g ph Agar 20g Soluble starch 10g, K2HPO4 1g,MgSO4.7H2O 1g,NaCl. 1g (NH4)2 SO4 2g CaCO3 2g Trace salt solution.1ml Distilled water 500ml Ph Oat meal 20g,Agar18 g, Trace salt solution 1ml Adjust ph

2 II Trace salt solution, colloidal chitin solution, and mercuric chloride solution SL. CHEMICALS Trace element solution (Peridham Gottlieb salt solution) Colloidal chitin (Hsu and Lockwood, 1956) Mercuric chloride solution COMPOSITION CuSO 4.5H 2 O 0.64g,FeSO 4.7H 2 O 0.11g, MnCl 2.4H 2 O 0.79g,ZnSO 4.7H 2 O 0.15g,Distilled water 100ml. Store at 3-5⁰C until required for use. Prepare fresh solution each month 5gm of chitin flakes with 60ml of HCl for 30min. Then cold distilled water was added to it. This precipitate was filtered. The filtered ppt. was again resuspended in cold distilled water. This process was repeated sveral times till the ph becomes 5-6. The final ppt was suspended in water and autoclaved. This can be stored upto several months for use. 15% Acidic mercuric chloride solution was prepared by weighing 15gms of Mercuric chloride in HCl 4. Potassium tetraborate 0.8M potassium tetraborate in water 5. DMAB reagent 6. DNSA reagent 5g DMAB IN 475ml glacial acetic acid and 6.25ml of 10N HCl 30g sodium potassium tartarate,2gm Sodium hydroxide, I gm Dinitrosalicylic acid powder. 152

3 III. Growth medium and solutions for biochemical tests used in the study SL. MEDIA COMPOSITION Colloidal chitin agar. Colloidal chitin 0.5%, K 2 HPO 4 0.7g, KH 2 PO 4 0.3g, MgSO 4 0.5gm, trace solution 1 ml Sea water LB medium Tryptone 5g, Yeast extract 5g, NaCl 5g, distilled water 500 ml, Sea water 500ml, ph 7 Basal mineral salt medium (NH4) 2 SO 4 64g, KH 2 PO 4 38g, K 2 HPO 4.3H 2 O 5.65g,MgSO 4.7H2O 0g, (Perdiham and Gottlieb, 1968) trace salt solution..1ml ph Agar 15g 4. Iodine solution 2gm Iodine and 1gm potassium iodide in 300ml distilled water. 5. Acidic mercury chloride 15gm HgCl2, 20ml HCl volume made upto 100ml by distilled water. 6. Congo red solution 1% w/v congored powder in water. IV Chemicals and Buffers used for DNA isolation, SDS- PAGE SL BUFFER COMPOSITION Potassium phosphate buffer ph 7 0.1M Mix 65ml of K 2 HPO 4 (1M) and 38.5ml of KH 2 PO 4 and make up the volume upto 10000ml with distilled water. 1M Tris Cl Buffer ph in1000ml ph adjusted by HCl 5 XTris Glycine 15.1g Tris base (25mM), 94g glycine Buffer (250mM), 50ml 10%SDS (0.1%) 4. 5 MTris cl ph g in 1000ml ph adjusted by HCl 5. 1M Tris Cl ph g in 1000ml ph adjusted by HCl 6. 1X TE 10mM Tris HCl, 1mMEDTA,pH 8 7. Lysozyme Buffer 8. CTAB/NaCl X PAGE loading buffer Staining solution for PAGE 25mM Tris HCl (ph 8), 25mM EDTA(pH 8), 300mM Sucrose 10% CTAB (Cetyltrimethylammonium bromide) in 0.7M NaCl (CTAB should be added slowly to NaCl at 65 C solution and autoclave 250mM Tris-Cl(pH 6.8), 500mM DTT,10% W/V SDS, 0.5%bromophenol blue, 50%(v/v) glycerol 0.25%(w/v) CBB R-250,45% (v/v) methanol, 10% (v/v) acetic acid in distilled water 153

4 1 Destaining solution 45% (v/v) methanol, 10 % (v/v) acetic acid 29.2gm of acrylamide and 0.8gm of bisacrylamide mixed. The volume was made Acrylamide solution 1 (30%) upto 100ml. V List of antibiotics used for antibiotic sensitivity test. SL REAGENT COMPOSITION PURPOSE 1 Aniline Diphenyl amine 2 Ninhydrin SL. ANTIBIOTIC CONCENTRATION 1 Amikacin 10mcg/disk 2 Ampicillin 3 Amoxycillin 50mcg/disk 4 Bacitracin 10unit/disk 5 Cotrimoxazole 25mcg/disk 6 Cloxacillin 30mcg/disk 7 Cephalexin 30mcg/disk 8 Carbenicillin 100mcg/disk 9 Ciprofloxacin 30mcg/disk 10 Cephacloxacin 30mcg/disk) 11 Erythromycin 10mcg/disk 12 Furazolidone 50mcg/disk 13 Kanamycin 30mcg/disk 14 Nitrofurantoin 300mcg/disk 15 Nalidixic acid 30mcg/disk 16 Norfloxain 10mcg/disk 17 Oxytetracyclin 30mcg/disk 18 Ofloxacin 10mcg/disk VI List of spray reagents used for TLC in this study 8gm aniline, 8gm diphenyl amine, 80ml acetone, 20ml Hydrochloric acid 0.2g Ninhydrin (Merck 6762) in100ml ethanol For analysis of oligosaccharide Polar lipid (amine group) 3 Molybdatophosphotic acid *5g molybdatophosphoric acid (Merck 532) in 100ml of ethanol 4 Zinzade reagent Readymade from Merck Total lipid (Phosphate)Polar lipid 154

5 5 Alpha naphthol 15ml of 15% l-naphthol (Merck 6223) solution in ethanol (w/v) with 85ml chloroform:methanol (25:30 v/v). 8.4ml of this solution is mixed with 5.2ml H2SO4, 32ml ethanol and 2 of H2O Appendix: II glycolipid I List of Instruments used in the current study S.. INSTRUMENT COMPANY Centrifuge Sigma, Germany Shaker incubator New Brunswick, Laminar air flow Sun Scientific, India 4. Vacuum evaporator Eyela, Japan. 5. SDS PAGE Gel Chromous electrophoresis biotech, India equipment 6. Gel documentation system UVP,USA 7. Water bath Julabo, 8. ph meter Sartorius 9. Microbalance Sartorius 10. Spectrophotometer Shimadzu, Japan 1 Transmission Carl Zeiss, 1 electron microscope Scanning electron microscope Germany Carl Zeiss Germany 155