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Appendix – I

MEDIA COMPOSITION

Christensen’s urea agar

Peptone - 1 g

Dextrose - 1 g

NaCl - 5 g

Disodium PO4 - 1.2 g

Monopotassium PO4 - 0.8 g

Phenol red - 0.012 g

Agar - 20 gm

pH - 6.8 0.2

40% Urea – after sodification

950 ml Dis.H2O + 50 ml sterile 40% urea solution.

Congo red medium

Yeast extract - 1 g

Mannitol - 10 g

Dipotassium phosphate - 0.5 g

Magnesium sulphate - 0.2 g

Sodium chloride - 0.1 g

Calcium carbonate - 1 g

Agar - 20 g

pH - 6.8 0.2

Congored - 2.5 ml of 1% solution/l.

Gelatin agar medium

Gelatin - 30 g

Casein - 10 g

NaCl - 10 g

Agar - 20 g

pH - 7.2 0.2

xxxvii Hofer’s alkaline broth

Dipotassium phosphate - 0.5 g, Yeast extract - 1g

Magnesium sulfate - 0.2 g, Mannitol - 10 g

Calcium carbonate - 0.05 g, pH - 11

Jensen’s medium

Calcium PO4 - 1 g

Dipotassium hydrogen PO4 - 0.2 g

Magnesium sulphate - 0.2 g

NaCl - 0.2 g

Ferric chloride - 0.1 g

Agar - 15 g

pH - 6.8

Lactose agar

Beef extract - 3 g

Peptone - 5 gm

Lactose - 5 g pH – 6.0 0.2

Agar - 20 g

Litmus milk agar

Litmus milk - litre

Yeast extract - 3 g

D-glucose - 10 gm

Agar - 20 gm

Mac-Conkey agar

Peptone - 17 gm

Protease - 3 gm

Inositol - 10 gm

Bilesalt - 1.5 gm

NaCl - 5 gm

Cry-violet - 0.001 gm

Neutral red - 0.03 gm

xxxviii

Agar - 20 gm

pH - 7.1 0.2

Modified phenol red broth

Protease peptone - 10 gm

Beef extract - 1 gm

NaCl - 5 gm

Phenol red - 0.025 gm

pH - 7.4 0.2

MR-VP broth

Peptone - 7 g

Dextrose - 5 g

Dipotassium - 5 g

pH - 6.9 0.2

Nitrate broth

Peptone - 5 g

Beef extract - 3 g

Potassium nitrate - 1 g

pH - 7 0.2

Simmon’s citrate agar

Magnesium sulphate - 0.2 g

Ammonium dihydrogen PO4 - 1 g

Dipotassium PO4 - 1 g

Sodium citrate - 2 g

NaCl - 5 g

Bromothymol blue - 0.08 g

Agar - 20 g

pH - 6.8 0.2

xxxix SIM agar

Pancreatic digest of casein - 20 g

Peptic digest of animal tissue - 6.1 g

Ferrous ammonium sulfate - 0.2 g

Sodium thiosulfate - 0.2 g

Agar - 3.5 g

Starch agar

Peptone - 5 g

NaCl - 5 g

Yeast extract - 1.5 g

Beef extract - 1.5 g

Starch (Soluble) - 2 g

Agar - 20 gm

pH - 7.4 0.2

Tryptone broth

Casein enzymic hydrolysate - 10 g

Sodium chloride - 5 g

pH - 7.5 0.2

Trypticas soy agar

Tryptone - 15 g

Soytone - 5 g

NaCl - 5 g

Agar - 15 g

pH - 6.8 0.2

Triple sugar iron agar

Peptone - 10 g

Casein - 10 g

Yeast extract - 3 g

Beef extract - 3 g

Lactose - 10 g

xl

Sucrose - 10 g

Dextrose - 10 g

NaCl - 5 g

FeSO4 - 0.2 g

Sodium thioSO4 - 0.3 g

Phenol red - 0.024 g

Agar - 20 gm

pH - 7.4 0.2

Yeast extract mannitol agar 1000 ml

Yeast extract - 1 g

Mannitol - 10 g

Dipotassium phosphate - 0.5 g

Magnesium sulphate - 0.2 g

Sodium chloride - 0.1 g

Calcium carbonate - 1 g

Agar - 20 g

pH - 6.8 0.2

Yeast extract mannitol broth

Yeast extract - 1 g

Mannitol - 10 g

Dipotassium phosphate - 0.5 g

Magnesium sulphate - 0.2 g

Sodium chloride - 0.1 g

Calcium carbonate - 1 g

pH - 6.8 0.2

xli

Appendix – II

REAGENT PREPARATION

Crystal violet

Crystal violet - 2.0 g

Ethyl alcohol (95%) - 20 ml

Gram’s iodine

Iodine - 1 gm

Potassium iodide - 2 g

Alcohol

Iodine - 5 gm

Potassium iodide - 2 g

Dis.H2O - 300 ml

Alcohol (decolourizer)

Iodine - 5 gm

Potassium iodide - 0.3 g

Methanol - 4.5 ml

Acetone - 20 ml

Safranine

Safranino - 2.5 gm

Ethylacohol (95%) - 100 ml

Carbol fuschin

Carbolfuchsin - 1 ml

Dis H2O - 19 ml

Benedict’s reagent

Copper - 18 g

Sodium carbonate - 100 g

Sodium citrate - 200 g

xlii

Potassium thiocyanate - 125 g

Potassium Ferricyanide - 250 mg

dis.H2O - 1000 ml

Methyl red solution

Suspend – 1g copper sulfate in 40 ml concentrated ammonia and

add 690 ml of an approximately 10 per cent potassium hydroxide solution.

Kovac’s reagent

Paradimethyl amino benzaldehyde - 5 gm

Amyl alcohol - 75 ml

HCl - 25 ml

Sulphonilic acid – 0.8%

Sulphanilic - 8 gm

30% acetic acid - 1000 ml

Alphanapthalamine solution

napthalamine - 5 gm

Acetic acid 5N - 1000 ml

Iodine solution

Iodine - 1 gm

Potassium iodine - 2 gm

dis. H2O - 300 ml

Barrit’s reagent

Suspend 5 g naphthol in 100 ml absolute ethanol.

Nutrient solution for plants in pot experiment

1. K2HPO4 - 0.2 g

2. (NH4)2 SO4 - 0.03 g

3. MgSO4 - 0.2 g

4. FeCl - 0.02 g

xliii 5. CaCl2 - 0.376 g

6. K2SO4 - 0.845 g

7. NH4NO3 - 0.1 g

8. H3PO3 - 1.855 mg

9. MnSO4 - 2.231 mg

10. ZnSO4 - 0.288 mg

11. CuSO4 - 0.25 mg

12. Na2M0O4 - 0.412 mg

Plasmid DNA isolation

Solution - I

Glucose 50 mM - 4.5 gm

Tris HCl 25 mM - 12.5 ml

EDTA 10 mM - 10 ml

pH - 8.0

Solution - II

NaOH 0.02 N

SDS 1%.

Solution - III

Potassium acetate - 5 m in 60 ml

Glacial acetic acid - 11.5 ml

Dis.H2O - 28.5 ml

pH - 4.8

TE buffer (pH 8.0)

Tris - 10 mM

EDTA - 10 Mm

ETBr

Dissolve 1 g of ETBr in 100 ml of dis.H2O, 8 stir with magnetic

bar for 1-2 hr.

xliv Electrophoresis buffer

Tris acetate buffer

Prepare 1 lit of 50X TAE, dissolve 242 g of trizma base, 57.1 ml of

glacial acetic acid and 100 ml of 0.5 M EDTA and adjust pH 8.

SDS-PAGE

1X SDS Gel loading buffer

Tris HCl (pH 6.8) - 1.2 ml

SDS - 1 gm

Glycerol - 3 ml

Bromophenol blue - 2 ml

Dis. H2O - 5 ml

Betamercaptoethanol - 200 ml

Add DTT from 1 M stock just before buffer usage.

1X Tris Glycine Electrophoresis buffer

Tris - 25 mM

Glycine (pH 8.3) - 250 mM

SDS - 0.1 %

Prepare 5X stock of electrophoresis buffer by dissolving 15.1 g Tris base,

94 g glycine in 900 ml of deionized H2O. Then add 50 ml of 10 per cent

(w/v) stock solution of electrophoresis grade SDS and adjust volume of 1

lit d.H2O.

Staining solution

B. Blue - 2.5 g

Methanol - 500 ml

Glacial acetic acid - 100 ml

Dis. H2O - 400 ml

Pilter it through whatman No:1 filter.

Destaining solution

Methanol - 500 ml

Glacial acetic acid - 100 ml

Dis. H2O - 400 ml

xlv Gel loading buffer

Bromo phenol blue - 0.25 %

Xylene Cyanol - 0.25%

Sucrose in H2O - 40%

TAE 1x

40 mM Tris acetate

1 mM EDTA.

TBE

45 mM Tris borate : 54 g Tris base

27.5 g boric acid

20 ml 0.5 M EDTA (pH 8.0)

SDS sample buffer : 2x

1 M Tris HCl (6.8) - 12.5 ml

SDS - 4 g

-mercaptoethanol - 10 ml

Glycerol - 20 ml

1% BPB - 4 ml

Add H2O to make final Vol to 100 ml.

Reservoir buffer

Tris 3 g

Glycine 14.4 g

SDS 1 g

adjust pH to 8.3 and make upto 1 l.

Reservoir gel

bis acrylamide - 12.5 gm

Resolving buffer - 3.75 (pH 8.8)

Stacking gel

2.5

5 (stacking buffer)

xlvi

10% SDS - 0.30 0.20

1.5% APS - 1.50 1.00

H2O - 11.95 11.30

TEMED - 0.015 0.015

30 ml 20 ml

Stackling gel buffer stock (Tris HCl, pH 6.8).

Staking gel buffer stock pH (6.8)

Tris 6.0 g

1 M HCl 48.0 ml

Adjust pH to 6.8 and make upto 100 ml H2O filter thro whatman Nol.1

filter.

Resolving gel buffer stock (pH 8.8)

Tris - 36.3 g

1 m HCl - 48 ml

pH - 8.8

make up to 100 ml filter whatman No.1 filter paper.

Protein Estimation Lowry Method

Reagent A

2% of Sodium carbonate in 0.1 N NaOH.

0.4 gm sodium hydroxide in 80 ml dis.H2O and then dissolve 2 gm

of sodium carbonate (anhydrous). Make up the volume to 100 ml dis.H2O

and stored at room temperature.

Reagent B

2% copper sulphate.

Dissolve 2 gm of copper sulfate to in 100 ml dis.H2O stored at

room temperature.

Reagent C

2% Sodium potassium tartarate or 2% trisodium citrate.

xlvii Dissolve 2 gm of sodium potassium tartarate or trisodium citrate in

100 ml dis.H2O stored at room temperature.

Alkaline copper sulfate (Reagent D)

Reagent A+B+C.

Mix 0.5 ml of reagent B and 0.5 ml of reagent C and then add 99 ml of

reagent A. If the reagent D becomes turbid do not use it (mix the reagent

is above order).

Folin’s phenol reagent

Diluted Folin’s phenol reagent 1:1 ratio with dis.H2O.

DNA estimation

Preparation of diphenyl amine reagent

Dissolve 1 gm of diphenyl amine in 97.5 ml of glacial acetic acid

and then add 2.5 ml con.H2SO4 mix well. Stored the reagent in brown

bottle of room temperature (Stable for 1 month).

DNA stock solution

Disolve standard DNA 2.2 mg/ml in 5 mM NaOH. Stored at 4°C

stable for atleast 6 month.