Appendix. Medium Composition. Peptone - 0.5gm (gram) Yeast extract - 0.5gm. Beef extract - 0.1gm. NaCl - 0.5g. Agar - 2gm. ph Starch - 0.

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1 Appendix Medium Composition Nutrient Agar Peptone - 0.5gm (gram) Yeast extract - 0.5gm Beef extract - 0.1gm NaCl - 0.5g Agar - 2gm Distilled water - 100ml ph Starch Agar Starch Peptone NaCl Yeast extract Agar Distilled water - 100ml ph - 7 Basal Medium Peptone - 0.6% MgSO % KCl % Starch - 1% Agar Distilled water - 100ml ph - 7

2 Production Media S.No Ingredients Weight 1 Lactose 1.5gm 2 Yeast extract 2gm 3 KH 2 PO gm 4 MnCl 2-4H 2 O gm 5 MgS 4. 7H 2 O 0.025gm 6 CaCl 2. 2H 2 O 0.005gm 7 FeSO4.7H 2 O 0.001gm 8 SDS 0.1gm 9 Distilled water 100ml 10 ph 7

3 Mass Cultivation Medium S.No Ingredients Weight 1 Lactose 1.5gm 2 Yeast extract 2gm 3 Casein 0.2gm 3 KH 2 PO gm 4 MnCl 2-4H 2 O gm 5 MgS 4. 7H 2 O 0.025gm 6 CaCl 2. 2H 2 O 0.005gm 7 FeSO4.7H 2 O 0.001gm 8 SDS 0.1gm 9 Distilled water 100ml 10 ph 7

4 Luria Bertani Broth Peptone - 1.0% gm Yeast extract - 0.5% gm NaCl - 1.0% gm Distilled water ml Buffers Sodium Phosphate Buffer A) Na 2 HPO g/l B) NaH 2 PO g/l Mix 51 ml of solution A and 49 ml of solution B make up to 200 ml with distilled water. Citrate Phosphate Buffer A) 0.2M Na 2 HPO ml B) 0.1M Citric acid ml Mix 10.3 ml of 0.2M Na 2 HPO 4 and 9.7 ml of 0.1M citric acid. TAE Buffer Tris HCL gm Glacial acetic acid ml 0.5 M EDTA - 10 ml Distilled water ml TE Buffer 10 mm Tris-HCL 1 mm EDTA

5 SDS - POLY ACRYLAMIDE GEL ELECTROPHORESIS REAGENTS 1. Acrylamide stock solution (monomer) A) Acrylamide - 30% B) N N -methylene bisacrylamide - 0.8% This is combination of 30 % acrylamide and methylene bisacrylamide. The initially dissolved in small amount and were making up to 25ml with glass distilled water. Monomer was stocked in amber bottle at 4 c. 2. TRIS Buffer (for to provide or maintain ph) Two buffer , ph8.8 (for separating gel) and 0.125M, ph6.8 (for stacking gel) were prepared and stored in brown bottle at 4 c % sodium dodecyl sulfate 4. 10% ammonium per sulfate 5. TEMED (Tetrramethyl Ethylene Diamine) Separating gel 0.375M Tris ( ph8.8) - 1.5ml Acrylamide - 2.0ml APS ml SDS ml Water ml TEMED ml Stacking gel 0.125M Tris ( ph6.8) ml Acrylamide - 1.5ml APS -.015ml

6 SDS ml Water - 1.7ml TEMED ml Sample Buffer 0.025M Tris (ph6.8) ml 2-mercoptoethnol - 0.1ml SDS ml Glycerol - 0.2ml Bromophenol blue % Tank Buffer ph 8.3 (0.025M Tris, 0.129M glycine and 10%SDS) Tris - 1.5gm Glycine - 7.2gm SDS - 0.5gm Water - 500µl Staining solution Coomassive brilliant blue mg Methanol - 80ml Glacial acetic acid - 20ml Distilled water - 100ml Genomic DNA isolation reagents CTAB (Cetyl Trimethyl Ammonium Bromide) NaCl TE buffer (10mM tris; 1 mm EDTA, ph 8.0). Lysozyme

7 Proteinase K 5M NaCl 10% SDS Chloroform Isoamyl alcohol Phenol Isopropanol Ethanol RNAse A (100mg/ml) PCR Mixture Sterile glass distilled water - 39µl 10X Assay buffer - 5µl 10mM dntps mix - 3µl Template DNA - 1µl Primer - 1µl Taq Polymerase - 1µl Total volume - 50µl DO Reagents used (g/lit) MnSO 4 4H 2 O Alkaline Iodide (g/lit) KOH KI Sulphuric acid concentrated - 5 ml Sodium thiosulfate (0.01N) Starch indicator - 1% Potassium dichromate (0.01N)

8 Nitrate reagents used 1. Concentrate ammonium chloride was dissolved in 500-ml 0f distilled water. 2. Diluted ammonium chloride: 5-ml of concentrated ammonium chloride solution were diluted to 200-ml with distilled water. 3. Sulphanilamide solution: 5-mg of sulphanilamide were dissolved in 50- ml of concentrated HCI and 300-mi distilled water and the volume was made up to 500-mi in the distilled water. 4. N-(1 napthyl) ethylene diamine di hydro chloride solution: 0.5-g of the salt was dissolved in 500-mi of distilled water. 5. Cadmium-copper column: It was prepared using metallic cadmium (99.9% Pure.) TWEEN 80 Agar (100ml) Peptone Beef extract Calcium Chloride g Tween ml NaCl - 0.5g Distilled water ml Agar g ph Oxdation and Fermentation basal medium-of Substrate - 7 g (Glucose, mannitol and fructose) Beef extract - g NaCl - 5 g Phenol red g

9 Distilled water ml ph Triple Sugar Iron Agar (g/lit) Peptone - 10 g Casien - 10 g Yeast extract - 3 g Beef extract - 3 g Lactose - 10 g Sucrose - 10 g Dextrose - 10 g Dextrose - 1 g NaCl - 5 g Ferrous sulphate h Sodium thiosulphate g Phenol red g Distilled water ml Agar - 12 g ph Trypticase Nitrate Broth (g/lit) Trypticase - 20 g Disodium phosphate - 2 g Dextrose - 1 g Distilled water ml Potassium nitrate - 1 g ph - 7.2

10 MR-VP Broth (g/lit) Peoptone - 7 g Dextrose - 5 g Dipotassium phoaphate - 5 g Distilled water ml ph Simmons Citrate Agar Ammonium di hydrogen phosphate - 1 g Dipotassium phosphate - 1 g NaCl - 5 g Magnesium sulfate g Agar - 15 g Bromothymol blue g Distilled water ml ph Commercial Feed Composition Protein supplements % of Crude Protein (CP) Energy Sources % of Crude Protein (CP) Fish meal 65% CP Ground corn 9% CP Soybean meal cake 45% CP Cassava flour 2% CP Total 110% CP Total 11% CP Average % CP Average % CP

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