appendices - shodhganga

Results and Discussion

Effect of tannery effluent on water and soil profile, plant growth and human health

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AAPPPPEENNDDIICCEESS

APPENDIX - 1

TOTAL HARDNESS EDTA titrimetric method (APHA, 2005)

PRINCIPLE

Total hardness is defined as the sum of the calcium and magnesium concentrations, both expressed as calcium carbonate in mg/l. EDTA and its sodium salts form a chelated soluble complex when added to a solution of certain metal cations. EDTA is added as a titrant, the calcium and magnesium will be complexed in the solution. It turns wine red to blue marking the end point of the titration. REAGENTS 1. Buffer solution: Dissolved 16.9g of ammonium chloride in 143ml ammonium hydroxide.

Added 1.25g magnesium salt of EDTA and diluted to 250ml with distilled water. 2. Indicator: Eriochrome black T: Dissolved 0.5g of dye in 100g of 2,2,2” nitrilotriethanol added

2 drops per 50mlsolution to be titrated. 3. Calmagite: Dissolved 0.1g of Calmagite in 100ml-distilled water. Used 1ml per 50 ml solution

to be titrated. 4. Standard EDTA titrant, 0.01M : Weighed 3.723g of analytical reagent grade EDTA,

dissolved in distilled water and diluted to 1000ml. Standardized against standard calcium solution.

5. Standard calcium solution: weighed 1.000g of anhydrous CaCO3 powder and diluted to 1000ml with distilled water.1ml=1.00mg CaCO3.

PROCEDURE

Diluted 25ml sample to about 50ml with distilled water in a suitable vessel. Added 1 to 2ml buffer solution. Usually 1ml will be sufficient to give a pH 10 to 10.1. Added 1 to 2 drops of indicator solution. Added EDTA titrant slowly, with continuous stirring, until the last reddish tinge disappears. Added the last few drops at 3 to 5 seconds intervals. At the end point the solution was normally blue. CALCULATION Hardness (EDTA) as mg CaCO3/ L=AxBx1000/ml sample. Where A= ml titration for sample B=mg CaCO3 equivalent to 1.00ml EDTA titrant.

APPENDIX – 2 ELECTRICAL CONDUCTIVITY

Conductivity method (APHA, 2005) PRINCIPLE

Conductivity is a numerical expression of the ability of an aqueous solution to carry an electric current. Conversely molecules of organic solution that do not dissociate in aqueous solution conduct a current poorly. REAGENTS 1. Conductivity water: Passed distilled water through a mixed bed deionize and discarded the

first litre. 2. Standard potassium chloride solution 0.0100M: Dissolved 745.6mg of anhydrous

potassium chloride (KCl) in conductivity water and diluted to 1000ml at 25ºC.



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PROCEDURE 1. Determination of cell constant: rinsed conductivity cell with at least three portions of 0.01M

KCl solution. Adjusted temperature to 25.0 ± 0.1ºC. Complete cell constant, C = (0.001413) (RkCl) (1+0.0191 (t-25) Where Rkcl=measured resistance ohms, t = observed temperature tºc.

2. Conductivity measurement: Rinsed cell with one or more portions of sample. Adjusted the temperature to 25.0 ± 0.1ºC.

CALCULATION K= (km) (c)/1+0.0019 (t-25), km=measured conductance mhos at tºC.

APPENDIX – 3 DETERMINATION OF TOTAL SUSPENDED SOLIDS

Filtration method (APHA, 2005) PRINCIPLE

A well-mixed sample is filtered through a weighed standard glass- fibre filter and the residue retained on the filter is dried to a constant weight at 103ºC–105ºC. The increase in weight of the filter represents the total suspended solids. If the suspended material clogs the filter and prolongs filtration, it may be necessary the diameter of the filter which decrease the sample volume. PROCEDURE

Filtered 250ml of the effluent through a tarred filter paper by applying suction. Washed the filter paper with distilled water to remove the soluble salts. Dried the filter paper for at least one hour in an oven at 103ºC –105ºC. The increase in weight was the total suspended solids. Expressed the result as mg total suspended solids per liter of the sample.

APPENDIX – 4 DETERMINATION OF TOTAL DISSOLVED SOLIDS

Filtraton method (APHA, 2005)

PROCEDURE Filtered 250ml of the effluent through a glass micro fiber filter paper. Evaporated the

filtrate in a tarred porcelain dish which was preheated at 105ºC and then at 550ºC for one hour in a muffle furnace, cooled and brought to constant weight. Kept the dish at 180ºC for about one hour, cooled and weighed. The increase in weight denoted the total dissolved solid content. Expressed the result as mg total dissolved solids per liter of the sample.

APPENDIX – 5 ESTIMATION OF CHEMICAL OXYGEN DEMAND

Titrimetric method (APHA, 2005)

PRINCIPLE Chemical oxygen demand (COD) is defined as the amount of a specified oxidant that

reacts with the sample under controlled conditions. The quantity of oxidant consumed is expressed in terms of its oxygen equivalence. Because of its unique chemical properties, the dichromate ion (Cr2O7)2 the specified oxidant is reduced to the chromate ion (Cr

3+).

COD often is used as a measurement of pollutants in wastewater and natural waters. Most types of organic matter are oxidized by boiling mixture of chromic and sulphuric acids. A sample is refluxed in strongly acid solution with a known excess of potassium dichromate. After digestion, the remaining unreduced potassium dichromate was titrated with features ammonium sulphate to determine the amount of potassium dichromate consumed and the oxidizable matter was calculated in terms of oxygen equivalent.



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REAGENTS 1. Mercuric sulphate crystals 2. Sulphuric acid- silver sulphate reagent: Dissolved 10.1g of silver sulphate in one litre of

concentrated sulphuric acid. Allowed the solution to stand for two days for complete dissolution.

3. Potassium dichromate solution 0.125N: Dissolved 0.129g in distilled water and made upto one litre. 1.0ml of 0.125N potassium dichromate = 1.0mg of oxygen.

4. Ferroin Indicator solution: Dissolved 95mg of ferrous sulphate in 500ml of distilled water. Added 1.48g of 1.10g phenanthroline monohydrate and mixed thoroughly.

5. Ferrous Ammonium Sulphate solution 0.125N: 40g of ferrous ammonium sulphate was dissolved in distilled water. Added 20ml of concentrated sulphuric acid. Made upto one litre with water. Standardized of it with 0.125N potassium dichromate.

PROCEDURE

A refluxing flask of 250ml capacity was used with a ground glass 24/40 neck fitted with a 300mm double surface condenser to which, a glass cap was fitted. Placed 50ml of the sample in the flask. Added mercuric sulphate of suitable quantity such that the ratio of chloride content of the sample to mercuric sulphate was 1:10 (For this, chloride content of the sample was estimated as given in appendix X). Then added 5ml of sulphuric acid-silver sulphate reagent and dissolved the mercuric sulphate. Cooled in cold water while mixing.

Pipetted 25ml of 0.125N potassium dichromate into the flask and mixed. Added a few

porcelain bits and attached the condenser. Started water circulation and refluxed for two hours. Removed the flame, allowed the flask to cool. Transferred the contents of the flask and diluted to about 350ml with distilled water. Added 2 to 3 drops of ferroin indicator and titrated against 0.125N ferrous ammonium sulphate solution. The end point was the sharp colour change from blue- green to reddish brown. A blank was conducted using 50ml of distilled water instead of the sample. CALCULATION

COD in mg/l = (blank titre value-sample titre value) x 0.125 x 1000 x 8 volume of the sample taken.

APPENDIX- 6

ESTIMATION OF BIOCHEMICAL OXYGEN DEMAND Winklers iodometric method (APHA, 2005)

PRINCIPLE

BOD determination involves the measurement of dissolved oxygen content of the sample, before and after 5 days incubation at 20ºC. The reduction in oxygen content to the demand exerted by the microbiological population and it is a measure of oxidizable organic matter in the sample. When manganous sulphate is added to the sample containing potassium oxide, magnesium hydroxide is formed, which is oxidized by the dissolved oxygen of the sample to basic manganic oxide on addition of sulphuric acid. The basic manganic oxide liberated iodine equivalent to the dissolved oxygen originally present in the sample. The liberated iodine was titrated with a standard solution of sodium thiosulphate using starch as indicator.

Reagents for the Preparation of dilution Water

1. Calcium chloride solution: 27.5g was dissolved in one litre of distilled water. 2. Magnesium sulphate solution: 0.25g was dissolved in one litre of distilled water. 3. Ferric chloride solution: 0.25g was dissolved in one litre of distilled water. 4. Phosphate buffer (pH7.2): 21.75g dipotassium hydrogen phosphate, 23.4g of disodium

hydrogen phosphate, 8.5g of potassium hydrogen phosphate and 1.7g of ammonium chloride, dissolved in 500ml of distilled water and make upto one litre with water.



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Reagents for the estimation of dissolved oxygen 1. Manganous Sulphate solution: Dissolved 91.0g of manganous sulphate in 250ml of distilled

water. 2. Alkali-iodide-azide reagent:

Reagent A : 175g of potassium hydroxide and 37.5g of potassium iodide were dissolved in 250ml water. Reagent B:2.5g sodium azide was dissolved in 10ml of water. Mixed reagent A and reagent B.

3. Concentrated sulphuric acid 4. Phosphoric acid: 80-90% 5. Sodium thiosulphate solution (0.1N): 24.82g was dissolved in one litre of distilled water. 6. Sodium thiosulphate solution (0.025N): Diluted 250ml of sodium thiosulphate solution

(0.1N) to 1000ml of distilled water.1.0ml of 0.025N sodium thiosulphate is equivalent to 0.2mg dissolved oxygen.

7. 1% starch solution. PROCEDURE Preparation of dilution water: Added 1.0ml of calcium chloride, magnesium sulphate, ferric chloride and phosphate buffer solutions to one litre of aerated distilled water and mixed thoroughly. This is the standard dilution water, prepared freshly every time.

Seeding of the dilution water: It is essential to seed the dilution water. The seeding material generally used is freshly settled raw sewage. 2.0ml of raw sewage was added to one litre of dilution water.

Dilution of the sample: The test water sample were diluted with seeded dilution water sample (1%, 5%,10%) in dilution mixture for the water sample. Each dilution sample was taken in a two set of BOD bottles.

Determination of dissolved oxygen (DO) before and after five days incubation: In one set of flasks DO was determined immediately while other set was kept for incubation at 20ºC for five days. DO of the incubated sample was determined.

Determination of DO is as follows: To the contents of the BOD bottle added 2.0ml of manganous sulphate solution and 2.0ml of alkali-iodide-azide solution. Stoppered the bottle and mixed thoroughly. A brown precipitate of basic manganic oxide was formed, which was allowed to settle completely leaving a clear supernatant liquid. Then added 2.0ml of concentrated sulphuric acid along the sides of the bottle. Stoppered and mixed for complete dissolution. Transferred the contents to a 500ml conical flask and titrated immediately against 0.025N sodium thiosulphate using starch as an indicator. CALCULATION FOR DO: Volume of 0.025N sodium thiosulphate used in the titration = DO in mg/l DO at 0ºC 760 mm pressure = DO x 0.07 mg/l

CALCULATION FOR BOD: BOD (5 days at 20ºC) = (DO0 - DO5 - BC) x100\percent sample. DO0 = Initial DO DO5 = DO after 20ºC incubation for 5 days BC = Blank correction i.e., Difference in DO of blank on the initial day and after 5 days incubation.

APPENDIX – 7 ESTIMATION OF CARBONATE AND BICARBONATE (ALKALINITY)

Titrimetric method (Natarajan et al., 1988) PRINCIPLE

When a sample containing carbonate and bicarbonate are titrated against the standard sulphuric acid, phenolphthalein lose its pink colour when half of the carbonate is converted into bicarbonate. So twice the value is a measure of carbonate present in the sample.



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To the colourless solution, a few drops of methyl orange is added and titrated against sulphuric acid till straw yellow colour changes to pinkish red colour. This value gives the amount of acid required to neutralize the carbonate originally present and that formed from the carbonates. By subtracting the first titre value from the second one, acid required to neutralize the bicarbonate originally present in the sample is obtained. REAGENTS 1. Sodium carbonate solution 1N: 1 3.25g was dissolved in 250ml water. 2. Sulphuric acid 1N: 28ml of concentrated sulphuric acid was made upto a liter with water. 3. Sulphuric acid 0.02N: Prepared from 1N sulphuric acid 4. Phenolphthalein indicator: 500mg was dissolved in 50ml of alcohol and 50ml of distilled

water. Added 0.02N sodium carbonate solution until a faint pink colour appeared. 5. Mixed indicator solution: 20 mg of methyl red and 100mg of bromocresol green were

dissolved in 100ml of 95% isopropyl alcohol. PROCEDURE

Added 25ml of the sample and 25ml of distilled water in a 250ml conical flask. Added phenolphthalein indicator solution. If no pink colouration , it indicated phenolphthalein alkalinity. If pink colour appeared then titrated with sulphuric acid (0.02N), until the solution became colourless. Added 3 drops of mixed indicator solution in which phenolphthalein alkalinity had been determined and titrated against sulphuric acid (0.02N) to light pink colour.

CALCULATION Volume of the sample taken = 25ml Volume of 0.1N sulphuric acid used up to phenolphthalein end point = A ml Volume of 0.1N sulphuric acid used up to methyl orange end point =B ml Volume of 0.1N sulphuric acid required up to neutralize bicarbonate alone = (B-A) ml. CARBONATE 1.0ml of 0.1N sulphuric acid=0.003g of CO3 2 x Aml of 0.1N sulphuric acid =0.003 x 2 x Ag Amount of carbonate per litre of sample=0.003 x 2 x 1000 x 1000/25m BICARBONATE 1.0ml of 0.1N sulphuric acid = 0.0061g of HCO3. (B-A)ml of 0.1N sulphuric acid = 0.0061x(B-A)g of HCO3 Amount of bicarbonate per litre of sample = 0.0061X(B-A) x 1000 x 1000/25mg

APPENDIX- 8 ESTIMATION OF CALCIUM

EDTA titrimetric method (APHA, 2005) PRINCIPLE

The pH of the sample is made sufficiently high (12-13) to precipitate magnesium as hydroxide and calcium only is allowed to react with EDTA in the presence of a selective indicator. REAGENTS 1. Sodium hydroxide 1N: 40g of sodium hydroxide was dissolved in one liter of distilled water. 2. Murexide indicator:200mg of the dye was ground with 100mg of sodium chloride. 3. Standard EDTA titrant, 0.02N. PROCEDURE

Pipetted out 50ml of the sample. Added 2.0ml of sodium hydroxide to it to produce a pH of 12- 13 and mixed well. Added 0.1-0.2g of the indicator, titrated immediately with EDTA. The end point is from pink to purple.



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CALCULATION

If the EDTA titrant is exactly 0.02N mg/l calcium (as CaCO3) = ml EDTA titrant x 1x 1000ml sample taken for the titration.

APPENDIX - 9

ESTIMATION OF MAGNESIUM Calculation method (APHA, 2005)

CALCULATION

Mg/l magnesium (as CaCO3) = mg/l total hardness (as CaCO3) = mg/l calcium (as CaCO3) mg/l.

APPENDIX – 10

ESTIMATION OF CHLORIDE Silver nitrate titrimetric method (Vogel, 1978)

PRINCIPLE

Silver nitrate reacts with chloride ions to form silver chloride. The completion of reaction is indicated by the red colour produced by the reaction of silver nitrate with potassium chromate solution, which is as added an indicator.

REAGENTS

Chloride free double distilled water was used for all the reagents 1. Standard silver nitrate titrant, 0.0282N: 4.791g of silver nitrate was dissolved in one litre of distilled water. Standardized it against 0.02N sodium chloride solution. 1.0ml of exactly 0.02N AgNO3 AgNO3 = 1.0mg of chloride. 2. Standard sodium chloride titrant, 0.0282N: 1.648g of sodium chloride was dissolved in one litre of distilled water 1.0ml=1.0mg of chloride. 3. Potassium chromate indicator solution: Dissolved 25g in 100ml of distilled water. Added silver nitrate solution drop wise until a slight red precipitate was formed. Allowed to stand for twelve hours. Filtered and made upto 500ml with distilled water.

4. Aluminium hydroxide suspension: Dissolved 100g of Aluminium ammonium sulphate in

1000ml distilled water. Warmed to 60ºC and added 55ml concentrated ammonia solution. Allowed the precipitate to settle for about an hour. Washed by decantation with distilled water to make the precipitate from chloride. Checked it by treating portion of the decanting every time with silver nitrate solution. After the precipitate was free from chloride, diluted it to 1000ml with distilled water.

PROCEDURE

Added 3.0ml of Aluminum hydroxide to a measured volume of the sample in a beaker. Stirred well and allowed to settle. Filtered, washed the precipitate with chloride free distilled water. Pipetted out 100ml of the sample into a porcelain dish. Adjusted the pH to be in the range of 7-9.5. Added 1ml of potassium chromate indicator solution. Titrated it against Standard silver nitrate solution with constant stirring until a slight precipitate reddish colouration persisted. Conducted a blank by placing 100ml chloride –free distilled water instead of sample.

CALCULATIONS

If the silver nitrate solution is exactly 0.0282N, Chloride mg/l = (ml AgNO3 for sample- ml AgNO3 for blank x1000 ml sample taken for estimation. If the silver nitrate solution is not exactly 0.0282N = (ml for AgNO3 sample- mlAgNO3 for blank) Chloride mg/l = Normality of AgNO3 x 35.45 x 1000ml sample taken for estimation.



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APPENDIX – 11 ESTIMATION OF SODIUM AND POTASSIUM

Flame photometric method (Natarajan et al., 1988) PRINCIPLE

In flame photometry, the solution under test is passed under carefully controlled conditions as a very fine spray in the air supply to a burner, in the flame the solution evaporates and the salt dissociates to give neutral atoms. A very small proportion of these moves into a higher energy state. When these excited atoms fall back to the ground state, the emitted of characteristic wavelength is measured. Sodium and potassium are measured at 590 and 770nm respectively. REAGENTS 1. Sodium stock solution: 2.524g of sodium chloride was dissolved in deionised water and

made upto one liter. 1.0ml = 1.0mg of sodium. 2. Potassium stock solution: 1.907g of potassium chloride was dissolved in deionised water

and made upto one liter. 1.0ml = 1.0mg of Potassium. PROCEDURE

Standardized the flame photometer before feeding the sample. Set the reading to zero using deionised water. Using the stock solutions of sodium and potassium, adjusted the reading to 100 at their specific wavelengths. Then feed the sample in the flame photometer and noted the readings to get the amounts of sodium and potassium directly on in milligrams per litre, by referring to the appropriate calibration curve.

APPENDIX-12 ESTIMATION OF FLUORIDE

Ion selective electrode method (APHA, 2005)

PRINCIPLE

The fluoride electrode is an ion-selective sensor. The fluoride electrode is the laser type doped lanthanum fluoride crystal across which a potential is established by fluoride solutions of different concentrations.

REAGENTS

1. Stock fluoride solution: Dissolved 221.0mg anhydrous sodium fluoride in 1000ml distilled water. 100ml=100 µg fluoride.

2. Standard fluoride solution: Diluted 100ml of stock solution to 1000ml with distilled water. 100ml =10.0µg fluoride.

3. Fluoride buffer: Placed approximately 500ml distilled water in a one liter beaker and added 57ml glacial acetic acid, 58g Nacl and 4.0g 1,2 EDTA. Placed the beaker in cool water bath and added slowly 6N NaOH, until the pH is between 5.3 and 5.5 make upto one litre with distilled water.

PROCEDURE

Prepared a series of standards by diluting standard fluoride solution 5.0, 10.0 and 20.0ml with 100ml distilled water, these standards are equivalent to 0.5, 1.0,and 2.0 mg fluoride/l. Added equal volume of fluoride buffer to samples were, standardized and immerse the electrode are measured.

CALCULATION

Mg fluoride/l= µg fluoride/ml sample



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APPENDIX – 13 ESTIMATION OF NITRATE

Nitrate electrode method (APHA, 2005)

REAGENTS

1. Nitrate free water 2. Stock nitrate solution: Dissolved 0.7218g of potassium nitrate in 1000ml of distilled water. 3. Standard nitrate solution: Diluted 1.0, 10 and 50ml stock nitrate solution to 100ml of water

to obtain standard solutions of 1.0, 10, and 50mg nitrate/L. 4. Buffer solution: Dissolved 17.32g aluminiumsulfate, 3.43g silver sulfate. Adjust the pH to 3.0

and diluted to 1000ml with distilled water. 5. Reference electrode filling solution: Dissolved 0.53g (NH4) 2SO4 in water and diluted to

100ml.

PROCEDURE

Transferred 10ml sample to a 50ml beaker, added 10ml buffer solution and stirred with a magnetic stirrer. Measured standards and samples at about the same temperature. Read concentration from calibration curve.

APPENDIX-14 ESTIMATION OF NITRITE

Colorimetric method (APHA, 2005)

PRINCIPLE

Nitrite is determined through formation of a reddish purple azo dye produced at pH 2.0 to 2.5 by coupling diazotized sulfanilamide with N- 1- naphthyl ethylenediamine dihydrochloride.

REAGENTS

1. Color reagent: To 800ml water, added 100ml 85% phosphoric acid and 10g sulfanilamide. After dissolving sulfanilamide completely, add 1g N- 1 - naphthyl ethylenediamine dihydrichloride. Mixed to dissolve and dilute to one litre with water.

2. Stock nitrite solution: Dissolved 1.232g sodium nitrite in 1000ml of distilled water. 3.Standard nitrite solution: Diluted 10.0ml nitrite solution to 1000ml with

water,1.00ml=0.500µg N.

PROCEDURE

Sample pH which was not between 5 and 9 was adjusted to that range. To 50.0ml of sample added 2.0ml of color reagent and mixed. After adding color reagent to standard and sample, the absorbance was measured at 543nm.

CALCULATION

Prepare a standard curve by plotting absorbance of standard against NO2-N concentration.

APPENDIX – 15 ESTIMATION OF SULPHATE

Turbidimetric method (APHA, 2005)

PRINCIPLE

Sulfate ion is precipitated in an acetic acid medium with barium chloride so as to form barium sulfate crystals. The sulfate concentration is determined by comparison of the reading with the standard curve.



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REAGENTS

1. Buffer solution: Dissolved 30g magnesium chloride, sodium acetate, 1.0g potassium nitrate and 20ml acetic acid in 500ml distilled water and made upto 1000ml.

2. Barium chloride, standard sulfate solution: Dissolved 0.1479g anhydrous sodium sulfate in distilled water and diluted to 1000ml.

PROCEDURE

Measured 100ml sample into an Erlenmeyer flask. Added 20ml buffer solution and mixed by stirring apparatus. Added a spoonful of barium chloride crystals. After stirring the readings are taken in a spectrophotometer at 420nm and measured turbidity at 5 ± 0.5 min.

APPENDIX - 16

ESTIMATION OF TOTAL CHROMIUM, ZINC, NICKEL AND CADMIUM Atomic absorption spectrophotometric method (APHA, 2005)

PRINCIPLE The technique involves determination of concentration of a substance by the measurement of absorption of characteristic radiation by the atomic vapour of an element. When radiation characteristic to a particular element passes through the atomic vapour of the same element, absorption of radiation occurs in proportion to the concentration of the atoms in the light path. The source of characteristic radiation is a hollow cathode lamp, the cathode being made of the element desired to be estimated.

REAGENTS

1. Air: Cleaned and dried through a suitable filter to remove oil, water and other foreign substances. The source may be a compressor or commercially bottled gas.

2. Acetylene, standard commercial grade. 3. Metal free water 4. Calcium solution: Dissolved 630mg of calcium carbonate in 50ml of 1+ hydrochloric

acid and diluted to 1000 ml with water. 5. Hydrochloric acid, 1%, 10%, 20% (all V/V), 1+5, 1+1 and concentrated. 6. Lanthanum solution: Dissolved 58.65g of lanthanum oxide in 250 ml conc.

Hydrochloric acid. Added slowly until the material was dissolved and diluted to 1000ml with water.

7. Hydrogen per oxide, 30% 8. Nitric acid, 2%(V/V), 1+1 and concentrated. 9. Aqua regia: Added 3 volumes concentrated HCl to one volume conc. Nitric acid. 10. Standard metal solutions: Prepared a series of standard metal solutions in the

optimum concentration range by appropriate dilution of the following stock solutions with water containing 1.5 ml conc. Niric acid/l

11. Chromium : Dissolved 0.1923g CrO3 in water acidified with 10ml Conc. Nitric acid and diluted to 1000ml with water. 1.0ml=100µg of chromium.

a) Cadmium: Weighed 100.0 mg pure Cd metal and dissolve in a solution composed of 20ml

water plus 5ml conc. HCL. Use heat to assist metal dissolution. Transfer quantitatively to a one liter volumetric flask and dilute to 1000ml

b) Standard Cadmium Solution : Pipetted 10.0ml stock solution into a volumetric flask, added 10ml conc. HCL and dilute to 1000ml with water. 1.0ml= 100µg cd.

c) Standard nickel sulphate solution : Dissolved 447.9 mg nickel sulphate in 1000ml distilled water.

d) Stock zinc solution: Dissolved 100mg 30-mesh zinc metal in a slight excess of 1+1 HCL; diluted to 1000ml with water. 1.00ml=100µg Zn



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Sample preparation 10ml of the effluent was taken in a 100ml kjeldahl flask. Added 25ml of 3:2:1 triple acid mixture (Conc. Nitric acid – Conc. Perchloric acid – Conc. Sulphuric acid) and left a side for 3-4 hours in a fume cupboard. Then heated for 30 min until the initial vigorous reaction has subsided. Heated more strongly for 4 hours until the nitrous fumes were removed and white fumes of perchloric acid were with 3-4 washings of deionised water to 10ml volumetric flask and made up to the mark with water. PROCEDURE Selected atleast three concentrations of each standard metal solution to find out the expected metal concentration of a sample. Then aspirated each standard in turn into flame and recorded the absorbance. Prepared a calibration curve by plotting the absorbance of standards versus their concentrations. Plotted calibration curve for chromium based on original concentration curve for chromium based on original concentration of standard before the addition of hydrogen peroxide. Rinsed nebulizer by aspirating. Water containing 1.5ml Conc. Nitric acid/l. Aspirated the sample and determined its absorbance against blank. The estimations of Chromium, copper and Zinc were done at the wavelengths of 357.9, 324.7, 248.3 and 213.9 nm respectively. CALCULATION

Calculated the concentration of each metal ion in milligrams per litre, by referring to the appropriate calibration curve.

APPENDIX -17 ESTIMATION OF NITROGEN

Titrimetric method (APHA, 2005)

REAGENTS

1. Mixed indicator solution: Dissolved 200mg of methyl red indicator in 100ml 95% ethyl alcohol. Dissolve 100mg methyeleneblue indicator in 100ml 95% ethyl alcohol.

2. Standard sulphuric acid titrant 0.02N

PROCEDURE

Titrated ammonia in distillate with standard 0.02N H2 SO4 titrant until indicator turns a pale lavender colour.

CALCULATION

Mg nitrogen/ l= (A-B) x 280/ml sample. A = volume of titrated H2 SO4 for sample, ml. B = volume of titrate H2 SO4 titrated for blank, ml.

APPENDIX - 18 ESTIMATION OF PHOSPHORUS

Spectrophotometry (Jackson, 1973) PRINCIPLE

Phosphorus reacts with ammonium molybdate and ammonium metavandate in acidic medium to give a yellow colour product. The intensity of the yellow colour was directly related to the concentration of phosphorus and was read at 420nm in a spectrophotometer.

REAGENT

1. Barteau reagent: Solution A : 25g of ammonium molybdate was dissolved in 400ml of warm water.

Solution B: 1.25g of ammonium metavandate was dissolved in 300ml of boiling water. Solution A and B were mixed and made up to 1000ml.



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2. Triple acid mixture: Concentrated nitric acid, perchloric acid and sulphuric acid were mixed in the ratio of 3:2:1.

PROCEDURE

5g of finely powdered sample was taken in a 100ml kjeldhal flask. Added 25ml of the triple acid mixture and heated for 4 hours, cooled and made up the solution to 100ml with distilled water. From the digested extract, pipetted out 5ml of the aliquot into a 25ml volumetric flask. Introduced a bit of red litmus paper. Neutralized with ammonia solution until litmus paper turned blue. Again neutralized with concentrated nitric acid until litmus paper turned red. Added 5ml of Barteau reagent. Made up the volume to 25ml with distilled water. After 30 minutes the intensity of yellow colour developed was read at 420nm in a spectrophotometer. Using the phosphorus standard curve, the phosphorus concentration in the sample was read in ppm.

Preparation of phosphorus standard curve

Analytical grade of potassium dihydrogen phosphate was dried at 40°C and 0.2195g was dissolved in 400ml of distilled water. 25ml of 7N sulphuric acid was added and volume was made up to 1000ml. This is 50ppm of phosphorus. Then 100ml of this 50 ppm stock solution was diluted to 100ml to get 5ppm of phosphorus solution. From this a series of phosphorus standard ranging from 0.001 ppm to 1ppm were prepared. Finally 5ml of this solution were pipetted out into a 25ml volumetric flask. 5ml of Barteau reagent was added and volume was made upto 25ml. Intensity of the colour of each standard was measured on the calorimeter and a standard curve was constructed using calorimeter readings and concentrations.

APPENDIX - 19 ESTIMATION OF COPPER

Colorimetric method (Raguramulu et al.,2003)

PRINCIPLE: Copper forms a coloured complex with sodium diethyl dithiocarbamate which is estimated colorimetrically. REAGENTS: 1. 2 N HCl 2. 20% TCA 3. Saturated solution of sodium pyrophosphate 4. Saturated solution of sodium citrate 5. Ammonium hydroxide diluted 2:1 with water 6. 0.1% solution of sodium diethyl dithiocarbamate. 7. Standard copper solution containing 100µg/ml : The standard is prepared by dissolving

392.8 mg of copper sulphate in distilled water and diluting the solution to l liter. Only crystals, which do not show efflorescence, should be used. Working standards are prepared from the stock solution to contain 0.8 µg and 2.0 µg/ml.

PROCEDURE: 1 ml of serum or plasma was taken and 1ml of 2 N HCl was added and allowed to stand for 10 min followed by the addition of 1 ml of 20% TCA. After stirring it was kept for 10 min and then centrifuged for 30min at 3000rpm. 2.4ml aliquot was collected and to it 0.2ml each of sodium pyrophosphate and sodium citrate were added, followed by the addition of 0.4ml of ammonium hydroxide. Glass distilled water was added to make up the volume to 3.3ml. The OD is read against a reagent blank containing no carbamate at 440nm. Added 0.2ml of sodium diethyl dithiocarbamate to each tube including the reagent blank. After mixing, it was kept for 15 min and the OD of the sample and blank were again read against the reagent blank containing no carbamate.



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CALCULATION: Plasma or serum copper (100µg/ml) = (D2 – D1)F x Kx 3.0 x 100

2.4 where,

D2 = OD after adding the carbamate D1 = OD before adding carnamate,

F = ratio between volume of the solution in the test tube before and after adding the carbamate K = constant to be determined from the standard graph

APPENDIX - 20 ESTIMATION OF IRON

DTPA method (Shanmugam et al., 1994) PRINCIPLE: Chelating agents combine with free metal ions in solution forming soluble complexes and there by reduce the activities of the free metal ions in solution. Therefore more metal ions desorb from the soil surface or liable solid phases to replenish the free metal ions in solution. REAGENTS: 1. The diethylene triamine penta acetic acid (DTPA) extracting solution was prepared to contian

0.005M DTPA, 0.01M CaCl2 0.1M TEA and pH adjusted to 7.3. To prepare 1 litre of this solution, dissolved 1.967g of DTPA, 1.47g CaCl2.2H2O, 13.3ml of reagent grade TEA in approximately 20ml of water. Allowed sufficient time for DTPA to dissolve and diluted to one litre. Adjusted the pH to 7.3+0.05 with 1:1 HCl.

2. Preparation of standard solution: 1.0g of Fe pure metal in 20ml of 1:1 HCl and diluted to 1 litre to 1000ppm or µg/ml. If FeSO4 .7H2O was used. 4.4g of FeSO4 .7H2O was dissolved in 1 liter to give 1000ppm of Fe.

PROCEDURE: 10g of air-dried soil was placed in a 50ml polyethylene bottle and 20 ml of DTPA extracting solution was added. The bottle was tightly closed with a stopper and shaken on a horizontal shaker for 2 hours. Then the suspension was filtered through Whatman No.42 filter paper. The filtrate was analysed for iron using atomic absorption spectrophotometer. Used the standard solution to prepare a calibration curve. Obtained the observation in ppm of element in the sample solution or on direct read out. CALCULATION:

Weight of soil taken = 10g Volume of DTPA extract added = 20ml Dilution = 2 times Concentration of the element in the sample =Sppm

solution as read forkm the standard curve

Concentration of element in the blank solution = Tppm Concentration of element in the soil = (S-T) x 2ppm.

APPENDIX -21

ESTIMATION OF CHLOROPHYLL (Yoshida et al., 1971)

PRINCIPLE Chlorophyll was extracted in 80% acetone and the absorption at 663nm and 645 nm

were read in a spectrophotometer. Using the absorption coefficient the amount of chlorophyll was calculated.



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REAGENTS 80% Acetone

PROCEDURE

1 gm of fresh leaves were cut into small pieces and homogenized in a mortor with pestle using 80% acetone. Decanted and filtered the supernatant through a funnel using Whatmann No.42 filter paper. Added sufficient quantity of 80% acetone and repeated the extraction. Transferred the contents from the motor to a funnel and washed the brei with acetone until it became colourless. Pooled the filtrates and made up the volume to 100 ml in a volumetric flask. Transferred, 5ml of the extract into a 50ml volumetric flask and diluted by making up the volume with 80% acetone. Measured the absorbance at 645 and 663 nm for the determination of chlorophyll-a and chlorophyll-b and total chlorophyll. The chlorophyll content was calculated on the fresh weight basis using the formula: mg chlorophyll a/g tissue=12.7(A663) -2.69(A645) X _ _ V______ 1000 X W

mg chlorophyll a/g tissue=22.9(A645) -4.68 (A663) X __ V______ 1000 X W

mg total chlorophyll b/g tissue=20.2 (A645) +8.02(A663) X ___ V______ 1000 X W

APPENDIX –22 ESTIMATION OF TOTAL CAROTENOID

(Zakaria et al., 1979)

PRINCIPLE

The total carotenoids in the sample were extracted in petroleum ether. The total carotenoids were estimated in UV/ visible spectrophotometer at 450 nm. REAGENTS 1. Petroleum ether 2. Anhydrous sodium sulphate 3. Calcium carbonate 4. 12% alcoholic KOH (ice cold) PROCEDURE Five to ten gram of the plant sample was weighed. After extracting the sample in 12% alcoholic KOH, the sample was saponified for about 30 minutes in a shaking water bath at 37˚C. The saponified sample was transferred into a separating funnel (packed with glass wool and calcium carbonate) containing 10-15 ml of petroleum ether and mixed gently. The petroleum ether layer with the carotenoid pigment was taken up. The lower aqueous phase was transformed into another separating funnel and the petroleum ether extract containing the carotenoid pigment to an amber coloured bottle. The extraction of the aqueous phase was repeated similarly with petroleum ether, until it becomes colourless. The aqueous phase was discarded. To the petroleum ether extract, added a small quantity of anhydrous sodium sulphate to remove turbidity. The final volume of the petroleum ether extract was noted and diluted if needed by a known dilution factor. The absorbance of the extract at 450 nm and 503 nm was noted in a spectrophotometer using petroleum ether as a blank. For total carotenoids Amount of total carotenoids present = P x 4 x V x 100µg W Where, P - Optical density of the sample, V - Volume of the sample, W - Weight of the sample



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APPENDIX-23 ESTIMATION OF CARBOHYDRATE

(Dubois, 1956)

PRINCIPLE

Carbohydrates were first hydrolysed into simple sugars using dilute hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxy methyl furfural. This forms a green coloured product with phenol and read colorimetrically at 490nm. REAGENTS 1. 2.5 N Hydrochloric acid 2. 95% Sulphuric acid 3. 0.2% Anthrone reagent: Dissolved 200mg of Anthrone in 100ml of 95% sulphuric acid. 4. Stock standard glucose solution: 100mg of glucose was dissolved in 100ml of distilled

water. 5. Working standard: 10ml of stock solution was diluted to 100ml with distilled water. PROCEDURE 100mg of the sample was weighed and it was hydrolysed by keeping it in a boiling water bath for 3 hours with 5ml of 2.5N hydrochloric acid and cooled to room temperature. They were neutralized with solid sodium carbonate until the effervescence ceases. The volume was made upto 100ml and centrifuged. 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard were pipetted out into a series of test tubes. 0.2ml of the sample solution was pipetted out into another test tube. The volume was made up to 1ml with distilled water in all the tubes. 1ml of water was served as blank. 4 ml of anthrone reagent was added, heated for 8 minutes in boiling water bath, cooled rapidly. The blue colour developed was read at 630 nm. APPENDIX – 24 ESTIMATION OF PROTEIN (Lowry et al., 1951) PRINCIPLE

The blue colour was developed by the reduction of the phosphomolybic phosphotungstic components. The Folin – Ciocalteau reagent by the amino acid tyrosine and tryptophan present in the protein plus the colour developed by the Biuret reaction of the protein with the alkaline cupric tartarate was measured in the Lowry’s method. REAGENTS 1. Reagent A: 2% sodium carbonate in 0.1N sodium hydroxide 2. Reagent B: 0.5%copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate. 3. Reagent C: Alkaline copper sulphate solution: Mixed 50ml of A and 1ml of B prior to use. 4. Reagent D: Folin-Ciocalteau Reagent: Refluxed gently for 10 hours a mixture consisting of

100g sodium tungstate, 25g sodium molybdate, 700ml water, 50ml of 85% phosphoric acid and 100ml of concentrated hydrochloric acid in a 1.5litre flask. Added 150g of lithium sulfate, 50ml water and a few drops of bromine water. Boiled the mixture for 15 min without condenser to remove excess bromine. Cooled, diluted to 1 litre and filtered.

5. Stock standard protein solution: Weighed accurately 50mg of bovine serum albumin and dissolved in distilled water and made up to 50ml in a standard flask.

6. Working standard: Diluted 10ml of the stock solution to 50ml with distilled water in a standard flask.

PROCEDURE Extraction of protein from sample

Weighed 500mg of the sample and grinded well with a mortar and pestle in 10ml of the phosphate buffer. Centrifuged and used the supernatant for protein estimation.



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Estimation of protein Pipetted out 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard into a series of test tubes. Pipetted out 0.1ml of the sample extract in another test tube. The volume was made up to 1ml in all the test tubes. A tube with 1ml of water served as a blank. Added 5ml of reagent C to all the test tubes including the blank. Mixed well and allowed to stand for 10 minutes then 0.5ml of reagent D was added, mixed well and incubated at room temperature in the dark for 30 minutes. Blue colour was developed. Readings were taken in spectrophotometer at 660nm. Standard graph was drawn and the amount of protein in the sample was calculated.

APPENDIX 25 ESTIMATION OF PHENOLS

(Malick and Singh, 1980) PRINCIPLE: Phenols react with phosphomolybdic acid in Folin- ciocalteau reagent in alkaline medium and produce blue coloured complex (Molybdenum blue) that can be estimated colorimetrically. REAGENTS: 80% ethanol, Folin- ciocalteau reagent, Sodium carbonate 20%. Standard 100mg catechol in 100ml water. Diluted 10times for working standard. PROCEDURE: 1. Weighed exactly 0.5g of the sample and ground with a pestle and mortar in 10 times volume

of 80% ethanol. 2. Centrifuged the homogenate at 10,000 rpm for 20min. Saved the supernatant reextracted with

5 times the volumes of 80% ethanol, centrifuged and pooled the supernatants. Evaporated the supernatant to dryness. Dissolved the residue in a known volume of distilled water (5ml). Pipetted out different aliquots (0.2 to 2ml) into test tubes made up the volume in each tube to 3ml with water. Added 0.5ml of folin- ciocalteau reagent. After 3 minutes, added 2ml of 20% Sodium carbonate solution to each tube. Mixed thoroughly and place the tubes in a boiling water bath exactly for one minute, cooled and measured the absorbance at 650nm against a reagent blank. Prepared a standard curve using different concentrations of catechol.

CALCULATION: From the standard curve concentrations of phenols in the test sample were found out and expressed as mg phenols/100g materi

APPENDIX – 26 POLYACRYLAMIDE GEL ELECTROPHORESIS

(Sambrook et al., 1989) Electrophoresis is widely used to separate and characterize proteins by applying electric current. The use of gels such as starch, polyacrylamide, agarose and agarose acrylamide as supporting media in electrophoretic techniques has enhanced resolution, particularly for proteins and aminoacids, due to a combination of reduced diffusion by the gel network and the separating action of gel chromatography.

Electrophoresis in acrylamide gels is frequently referred as polyacrylamide gel electrophoresis. Cross-linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of smaller amounts of bisacrylamide. Acrylamide monomer is polymerized in a head tail fashion into a long chain, which is coss-linked by bisacrylamide to form a matrix.

AIM : To electrophoretically separate the proteins in the given sample.



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REAGENTS

1. Acrylamide stock solution : Acrylamide: 29g, Bisacrylamide :1.0g, Distilled water :100ml 2. Resolving gel : Acrylamide stock : 2ml, Water : 1.6ml, Tris-HCl (p

H 8.8) : 1.3ml, 10% SDS:

.05ml, 10% APS : 0.05ml, TEMED : 20µl 3. Stacking gel : Acrylamide stock : 0.5ml, Water : 2.1ml, Tris-HCl (p

H 6.8) : 0.38ml,

10% SDS : 0.03ml, 10% APS,: 0.03ml, TEMED: 10µl, 4. Electrophoretic buffer 5X : Tris HCl (25mM) :0.003ml, Glycine (25mM) :0.018ml, Adjust

the pH to 8.3 and add 0.1% SDS

5. Loading Dye 1X : Tris HCl 6.8:50Mm, Beta mercaptoethanol :100mM, SDS,: 2%, Bromophenol blue: 0.1%, Glycerol : 10%

6.Staining solution:Coomasie Brilliant Blue R250: 0.25g, Methanol:90ml, Distilled water: 90ml, Glacial acetic acid: 20ml,

7. Destaining solution : Methanol :90ml, Distilled water : 90ml, Glacial acetic acid : 20ml PROCEDURE: Cleaned the glass plates, spacers and combs very well with ethanol and it was dried. Applied silicons around the edges of the spacers and holded them in place to seal the chamber in the glass plates. It was assembled properly using clips and clamped it onto the right position. Prepared sufficient volume of 12% resolving gel mixture. It was poured it into the space between the glass plates with out any air bubbles and layed distilled water on top of the gel. It was left to polymerise for 30- 60 minutes.

Discarded the laid over distilled water on top of the gel after polymerisation of the resolving gel. Prepared about 3ml of 5% stacking gel and poured them over the resolving gel and placed the combs in the same and allowed it to polymerize. On polymerization of the stacking gel the comb was removed without distorting the shape of the wells. The glass plates were fitted in the electrophoretic apparatus after removing the spacers at the bottom. The electrophoretic buffer was poured without forming air bubbles at the bottom of the gel. The cathode was connected at the top and the anode at the bottom and the power was turn on after loading the protein sample into the wells. The samples were prepared by taking equal concentrations of proteins in each well and mixed it with loading buffer, boiled it for a minute and after cooling, it was used for loading. The current was put off when the dye-front reached 1cm above the bottom of the gel. Removed the gel and carefully it was immersed in staining solution for 2-3 hours, the proteins absorb the stain. Then the gel was transferred to suitable container with atleast 200-300ml destaining solution and shook gently overnight. Photographed the bands that appear on the gel.

APPENDIX-27

ASSAY OF CATALASE ACTIVITY (Luck, 1974)

The UV light absorption of hydrogen peroxide can be easily measured between 230 and

250 nm. On decomposition of hydrogen peroxide by catalase, the absorption decrease with time. The enzyme activity can be arrived at from this decrease. REAGENTS 1. Phosphate Buffer: 0.067 M (pH 7.0): Dissolved 3.522g of KH2PO4 and 7.268g of Na2HPO4.2H2O in distilled water and made up the volume to one liter. 2. Hydrogen peroxide: Phosphate buffer: Diluted 0.16 ml of H2O2(10%W/V) to 100ml with phosphate buffer, prepared freshly. The absorbance of the solution should be about 0.5 at 240 nm with a 1cm light path. PROCEDURE ENZYME EXTRACT The homogenate of the incubated liver slices was employed for the assay.



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ASSAY Read against a control cuvette but containing the enzyme solution as in the

experimental cuvette but containing H2O2 phosphate buffer. Mixed in 0.01-0.04ml sample with a glass/plastic rod flattened at one end. Noted the time ‘t ‘ required for a decrease in absorbance from 0.45 to 0.4. This value was used for the calculations. If ‘t’was more than 60 seconds, then repealed the measurements with a more concentrated solution of the sample. CALCULATIONS Calculated the concentration of H2O2 using the extinction coefficient 0.036 per µ mole per ml. APPENDIX – 28 ESTIMATION OF PEROXIDASE ACTIVITY (Reddy et al., 1995) PRINCIPLE

In the presence of a hydrogen donor (pyrogallol or dianisidine) peroxidase converts H2O2 to water and oxygen. The oxidation of pyrogallol to colored product called purpurogalli is followed colorimetrically. Pyrogallol + H2O2 → Oxidised Pyrogallol + H2O+ O2 (coloured) REAGENTS 1. Pyrogallol 0.05M in 0.1M phosphate buffer (pH 6.5) 2. 1% H2O2

3. Enzyme extract Macerated one part of plant tissue with 5 parts (W/V) of 0.1M phosphate buffer (pH6.5) in a

homogenizer, centrifuged the homogenate at 500g for 15 minutes, used the supernatents is the enzyme source. All procedures were carried out at 0-5°C.

PROCEDURE

Pipetted out 3ml of 0.05M pyrogallol solution and 0.02ml of enzyme extract in a test tube. Adjusted the spectrophotometer to read ‘0’ at 430nm. Added 0.5ml of 1% H2O2 in the cuvette. Recorded the change in absorbance for every 30 seconds up to 3 minutes. CALCULATIONS: Change in absorbance/minute at 430nm = X Weight of the plant material taken = 300mg Volume of the sample taken for the assay = 0.2ml Change in absorbance for 0.02ml = X Change in absorbance for 1.5ml extract = (X/0.02) x 1.50 =Y (i.e) Peroxidase activity in 300mg plant tissue = Y Peroxidase activity /gram of plant tissue = Y x (1000/300) Units

APPENDIX -29 ASSAY OF SUPEROXIDE DISMUTASE (Kakkar et al., 1984)

The assay of SOD was based on the inhibition of formation of NADH phenazine methosulphate-nitroblue tetrazolium formazon. The reaction was initiated by the addition of NADH. After incubation for 90 seconds, the reaction was stopped by the addition of glacial acetic acid. The colour formed at the end of the reaction was extracted into butanol layer and measured at 560nm. REAGENTS 1. Sodium pyrophosphate buffer-0.025M pH 8.3: 1.115 g of disodium pyrophosphate was dissolved in 100ml of distilled water, and the pH of the buffer was adjusted to 8.3 by using 0.025M HCl.



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2. Phenazine methosulphate (186 µmol): 6mg in 100ml of distilled water 3. Nitroblue tetrazolium(300 µmol) 24.5 mg of nitroblue tetrazolium was dissolved in 100ml distilled water. 4. NADH (780 µmol): 51.8 mg of NADH was dissolved in 100ml distilled water. 5. Glacial acetic acid 6. n-butanol 7. Chloroform 8. Ethanol PROCEDURE

To 0.4 ml of the sample (mitochondrial and exmitochondrial fractions from rat liver homogenate), added 1.0 ml of ethanol and 0.6 ml of chloroform. This mixture was shaken for one minute at 4°C and then centrifuged. The enzyme activity in the supernatant was determined. The assay mixture contained 1.2ml of sodium pyrophosphate buffer, 0.1ml of phenazine methosulphate, 0.3 ml of nitroblue tetrazolium, approximately diluted enzyme preparation and HBSS in a total volume of 3.0 ml. The reaction was started by the addition of 0.2 ml of NADH.

After incubation at 30°C for 90seconds, the reaction was stopped by the addition of 1.0 ml of glacial acetic acid. The reaction mixture was stirred vigorously and shaken with 4.0 ml of n-butanol. The mixture was allowed to stand for 10 minutes and then centrifuged at 2000 rpm for 5 minutes. The intensity of the chromogen in butanol layer was measured at 560 nm against butanol as blank and the system devoid of enzyme served as control. One unit of enzyme activity is defined as the enzyme reaction which gave 50 % inhibition of NBT reduction in one minute under the assay conditions and expressed as specific activity in unit.

APPENDIX - 30 ESTIMATION OF GLUTATHIONE REDUCTASE

Spectrophotometric Method (Glatzle et al., 1970) PRINCIPLE: The enzyme glutathione reductase catalyses the reaction of oxidised glutathione to reduced glutathione.

GSSG+ NADPH + H+ FAD

2GSH + NADP+

The decrease in the NADPH concentration which is proportional to the enzyme activity

was measured. REAGENTS: 1. Phosphate Buffer pH 7.4: A. Dissolved KH2 PO4 2.722g in distilled water and made up to 200ml. Stored at 4°C B. Dissolved KH2PO4.3H2O. 22.8g in distilled water and made up to l litre. Stored at 4°C The two components were allowed to reach room temperature. Poured 100ml of component

‘B’ into a beaker and mixed component ‘A’ to get a pH 7.4. 2. 0.1 M sodium bicarbonate : Dissolved 8.4g NaHCO3 in distilled water and volume to 1 litre. 3. 50mM GSSG solution: Weighed 155mg of GSSG into a small beaker. Added 5ml of distilled

water and 50 µl of 0.8 M NaOH. Mixed well. 4. 250 µM Flavin Adenine Dinucleotide (FAD) solution: Weighed 1.0g of FAD into a small

beaker, added 5ml of distilled water. Mixed well. 5. 4mM NADPH solution: Weighed 33.2 mg of NADPH into a small beaker. Added 10ml of 1%

sodium bicarbonate solution. Mixed well. 6. 80 mM EDTA solution: Potassium EDTA- 3.2g in distilled water and made up to 100ml. 7. Preparation of hemolysate: To 200 µl of the frozen red cells added 3.0ml of phosphate buffer.

Mixed well and centrifuged. PROCEDURE: Tubes were set up as follows:



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Reagents Tube I (ml) Tube II (ml) Phosphate Buffer 2.0 2.10 Hemolysate 0.10 0.10 GSSG 0.10 0.10 FAD 0.10 - EDTA 0.05 0.05 The tubes were incubated at 37°C for 8 minutes. Following the initial incubation, the reaction was started by the addition of NADPH. With this assay it was shown that a blank without enzyme was not required for incubation period used.

The reduction of the absorbance, (oxidation) of NADPH was followed for 10 minutes at 37°C. The samples with and without FAD were always measured simultaneously in the same instrument. The coefficient was determined by dividing amount of reduction of absorbance of the assay with added FAD/10 minutes. The assay was linear for atleast 10 minutes.

APPENDIX-31

ESTIMATION OF ASCORBIC ACID (Roe and Keuther, 1953 )

PRINCIPLE Ascorbate is converted to dehydroascorbate by treatment with activated charcoal or

bromine. Dehydroascorbic acid then reacts with 2, 4 – dinitrophenylhydrazine to form osazones, which dissolved in sulphuric acid to give an orange coloured solution whose absorbance can be measured spectrophotometrically at 540 nm. REAGENTS 1. 4% Trichloro acetic acid 2. 9 N Sulphuric acid 3. 2% 2, 4 – dinitrophenylthydrzine reagent (DNPH) 4. 10% Thiourea 5. 85% Sulphuric acid 6. Stock standard solution: Dissolved 100 mg of ascorbic acid in 100 ml of 4% TCA 7. Working standard solution: Diluted 10ml of stock solution to 100 ml with 4% TCA PROCEDURE

Ground 1 g of the sample and homogenized in 4% TCA. Made up to 10ml and centrifuged at 2000rpm for 10 minutes. The supernatant obtained was treated with a pinch of activated charcoal, shaken well and kept for 10 minutes. Centrifuged once again to remove the charcoal residue. Noted the volume of clear supernatant obtained. 0.5 and 1 ml aliquots of this supernatant were taken for the assay. The assay volume was made upto 2.0 ml with 4% TCA. 0.2 to 1.0 ml of working standard solution containing 20-100 µg of ascorbate respectively was pipette out into clean dry test tubes and the volume was made up to 2.0 ml with 4% TCA. Added 0.5 ml of DNPH reagent to all the tubes followed by 2 drops of 10% thiourea solution . Incubated at 37°C for 3 hours. The oszone formed was dissolved in 2.5 ml of 85% sulphuric acid, in cold, drop by drop with no appreciable rise in temperature. To the blank alone, DNPH reagent and thiourea were added after the addition of sulphuric acid. After incubation for 30 minutes at room temperature, the absorbance was read spectrophotometrically at 540 nm.

APPENDIX- 32

ESTIMATION OF RIBOFLAVIN (Raghuramalu et al., 2003)

PRINCIPLE:

The native fluorescence of riboflavin in neutral pH is used in the chemical estimation of this vitamin.



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REAGENTS: 1. Riboflavin standard solution: 25mg of riboflavin is dissolved in 300 to 400ml of water, adding 1.2ml of glacial acetic acid and warming at a low temperature. After the riboflavin is dissolved, the solution is cooled and made up to 1 litre. This stock solution has a concentration of 25 µg /ml. 2ml of this stock solution is diluted to 5ml to give a working standard of 1µg /ml 2. 4% potassium permanganate. 3. 1:1 H2O2- water mixture PROCEDURE: To about 25ml of the vitamin extract, one or two drops of caprylic alcohol was added, followed by 3ml of freshly prepared 4% potassium permanganate solution. The mixture was stirred well and within 2minute, 3ml of 1:1 H2O2- water solution was added to discharge the permanganate colour and the pH adjusted to 7.0 with NaOH. The volume was made upto 35ml and the solution was filtered and the fluorescence of the filtrate was measured in a fluorimeter using the appropriate filters. CALCULTION: The fluorescence of a known aliquot of the filtrate as A µg of riboflavin was added and the reading noted as B. A small pinch of sodium hydrosulphite was added to destroy the riboflavin only and the reading recorded as C. Then the quantity of riboflavin in the solution taking for fluorimetry = A-B x 1 µg. B-C From this, the riboflavin content was calculated making due allowance for dilution and enzyme blanks etc.

APPENDIX - 33

ESTIMATION OF FLAVONOIDS (Cameron et al., 1943)

EXTRACTION: A portion of the ground plant material was weighed out and extraction was carried out in two steps, firstly with MeOH: H2O (9:1) and secondly MeOH: H2O (1:1). At each step, sufficient solvent was added to make liquid slurry and the mixture was left for 6-12 hours. Filtration to separate the extract from the plant material was carried out rapidly by using a glass wool or cotton wool plug in the neck of a filter funnel. The two extracts were then combined and evaporated to about 1/3 rd of the original volume or until most of the MeOH had been removed. The resultant aqueous extract was cleared of low polarity contaminants such as fats, terpenes, chlorophylls and xanthophylls by extraction (in a separating funnel) with hexane or chloroform. This was repeated several times and the extracts combined. The solvent – extracted aqueous layer containing the bulk of the flavonoids was then concentrated. REAGENTS: 1. Vanillin reagent- 1% vanillin in 70% concentrated H2 SO4 2. Catechin standard – (110 µg/ml) PROCEDURE: An aliquot of the extract was pipetted into a test tube and evaporated to dryness. Then added 4ml of vannilin reagent and heated for 15 minutes in a boiling water bath. A standard was also treated in the same manner. Then the optical density was read at 340 or 360nm.

APPENDIX -34

ESTIMATION OF TOCOPHEROL (Varley et al., 1991) PRINCIPLE:

Tocopherol can be estimated using Emmeric- Engle reaction which based on the reduction of ferric to ferrous ions by tocopherols, which then forms a red colour with 2,2’- dipyridyl-tocopherol and carotenes are first extracted with xylene and the extraction read at



222

460nm to measure carotenes. A correlation is made for these after adding ferric chloride and reading at 520nm.

REAGENTS: 1. Absolute alcohol

2. Xylene. 3. 2,2’- dipyridyl 4. Ferric chloride solution.

5. Standard solution: Dissolved 10mg/ 1 of tocopherol in absolute alcohol. 91mg of tocopherol is equivalent to 100mg of tocopherol acetate.

EXTRACTION:

The sample was homogenized with water in a blender. Weighed accurately, 2.5g of the homogenized sample into a conical flask. Added 50ml of 0.1 N H2 SO4 slowly without shaking. Stoppered and allowed to stand overnight. The next day, contents of the flask were shaken vigorously and filtered through whatman No.1 filter paper, discarding the initial 10-15ml of filtrate. Aliquots of the filtrate were used for the estimation.

PROCEDURE: Into 3 stoppered centrifuge tubes (test, standard and blank), pipetted out 1.5ml of extract, 1.5ml of standard, 1.5ml of water respectively to the test and blank added 1.5ml of ethanol and to the standard, added 1.5ml of water. Added 1.5ml of xylene to the test tubes, stoppered, mixed well and centrifuged. Transferred 1.0ml of xylene layer into another stoppered tube, taking care not to include any other ethanol or protein. Added 1.0ml of 2,2’- dipyridyl reagent to each tube, stoppered and mixed. Pipetted out 1.5ml of the mixture into colorimeter cuvettes and read the extinction of the test and standard against the blank at 460nm. Then in turn beginning with the blank, added 0.33ml of ferric chloride solution. Mixed well and after 15minutes read tests and standard against blank at 520nm. The amount of vitamin E can be calculated using the formula, Amount of tocopherols = Reading at 520nm-Reading at 460nm x 0.29 x 15 Reading at 520

APPENDIX - 35

HISTOCHEMICAL STAINING Silver Sulphide Method of Heavy Metals (Timm, 1958).

Preparation of the developer solution:

1. Gum Arabic (20%), allow it to stand for 7-14 days (100ml) 2. 10% AgNO3 – freshly prepared (1ml) 3. 5g citric acid and 2g hydroquinone in 100ml (10ml)

Mixed the three solutions in amounts stated in brackets immediately before use.

PROCEDURE: 1. Fixed blocks in 70% ethanol, saturated with H2S and made alkaline with 2 drops / 100ml

of concentrated NH3. Leave for 8-12 hours at room temperature. Take hand sections of the leaf, petiole and root.

2. Brought sections to water. 3. Treated with developer solution in the dark at 22˚C for 20-60 minutes (until sections are

slightly brown). Up to 6 hours development is necessary. 4. Washed in distilled water 5. Counterstained in 2% safranine for 2 minutes 6. Dehydrated , cleared and mounted in DPX mounted



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APPENDIX - 36 ESTIMATION OF HEMOGLOBIN

Cyanmethemoglobin method (Wintrobe et al., 1965) PRINCIPLE: Hemoglobin is oxidized by potassium ferricyanide to methemoglobin, which is then converted to cyanmethemoglobin by potassium cyanide. The intensity of colour produced by the formation of cyanmethemoglobin is directly proportional to the hemoglobin concentration and is measured photometrically at 540nm or with green filter. MATERIALS: 1. Drabkin solution : Dissolved 0.05g of potassium cyanide, 0.2g of potassium ferricyanide

and 1.0g of sodium bicarbonate in 1 liter of distilled water. 2. Cyanmethemoglobin standard: Supplied in sealed ampoules of 60mg per 100ml. PROCEDURE: Transferred 0.02ml blood with the help of hemoglobin pipette into a test tube containing 5.0ml of Drabkin’s solution. The tubes were mixed and the readings were taken in a spectrophotometer at 540nm. The reagent blank (Drabkin’s solution) was adjusted to zero. The standard was read in the same way after adding Drabkin’s solution. CALCULATION: Grams hemoglobin per 100ml blood = Reading of unknown x Dilution factor x Concentration of standard Reading of standard 1000

APPENDIX - 37 ENUMERATION OF TOTAL LEUKOCYTE COUNT

(Raghuramulu et.al., 2003) PRINCIPLE: Leucocyte being less in number, a dilution of only 1 to 20 is used and the diluent is usually one which destroys the red cells. MATERIALS: White blood corpuscles diluting fluid was prepared by mixing, 1. Glacial acetic acid : 3ml 2. Gentian violet 1% : Added till it gives a pale violet colour. 3. Water : 97ml PROCEDURE: The method of counting is similar to that of the red blood corpuscles except that the count is made in four large (1mm) corner square of the Neubauer counting chamber. CALCULATION: The total number of cells in four squares is multiplied by a factor of 2500 to give the count / mm

3 of blood.

APPENDIX - 38

ESTIMATION OF SERUM IMMUNOGLOBIN E (IgE) QUORUM EIA Ferritin Method (Nakamura et al., 1991)

PRINCIPLE: Specific monoclonal anti- IgE antibodies are coated on to microtitration wells. Test sera are applied and incubated with the zero buffer. If human IgE is present in the sample, it will combine with the antibody on the well. The well is then washed to remove any residue test specimen and then, IgE antibody labelled with Horseradish Peroxidase enzyme (conjugate) is



224

added. This results in the IgE molecules being sandwiched between the solid phase and the enzyme linked antibodies. After a 30 minute incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. On addition of the substrate ( TMB), a colour will develop only in those wells in which enzyme is present, indicating the presence on IgE. The enzyme reaction is stopped by the addition of hydrochloric acid and the absorbance is then measured at 450nm. The concentration of IgE is directly proportional to the colour intensity of the test sample. REAGENTS:

1. Reference standard set containing: 0, 10, 50, 100, 400 and 800 IU/ml IgE 2. Anti IgE- HRP conjugate working strength 3. Substrate solution (TMB): 2.08g of 3,3’5,5’- teramethyl benzidine (TMB) was dissolved

in 18.0 ml of glacial acetic acid. 100 ml of 3% hydrogen peroxide was added and made up the volume to 200ml with distilled water

4. Stock solution (2N hydrochloric acid) 5. Zero buffer (Phosphate buffer 0.1M, pH7.0):To 39.0ml of 0.2M solution of monobasic

sodium phosphate (27.8g in 1000 ml) added 61.0ml of 0.2 M solution of dibasic sodium phosphate (53.65 g in 1000ml) and diluted to a total of 200ml with distilled water

MATERIALS

1. Microtitration plate: wells coated with specific antibody 2. Micropipette 3. Distilled water 4. Microplate reader fitted with 450nm filter

PROCEDURE: 1. 20µL of serum samples and standard were added to the respective wells, followed by

100µL of zero buffer into each well. Mixed gently for 10 seconds. 2. Incubated of 30 minutes at room temperature. 3. Discarded the well contents and washed 5 times. 4. 150µL of enzyme conjugate was added into each well and mixed for 5 seconds. 5. Incubated for 30 minutes at room temperature. 6. Discarded the well contents and washed 5 times with distilled water. 7. Then added 200µL of substrate solution into each well and mixed for 5 seconds. 8. Incubated in the dark for 20 minutes at room temperature. 9. 50µL of stop solution (2N hydrochloric acid) was added to each well and mixed for 30

seconds. 10. The optical densities were read with an EIA reader using a 450nm filter.

CALCULATION:

A standard graph was drawn by plotting the optical density of each standard at 450nm on the Y-axis against its IgE concentration on the X-axis on a graph. The optical density of each specimen was plotted on the graph and the corresponding IgE concentration was found out.

APPENDIX- 39 ESTIMATION OF ALANINE TRANSAMINASE (SGPT)

(Reitman and Frankel, 1957) PRINCIPLE: SGPT catalyses the transfer of an amino group from alanine to α-Ketoglutarate with the formation of pyruvate and glutamate.

SGPT

Alanine + α-Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH+H

+ Lactate + NAD

+



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It catalyses the reduction of pyruvate to lactate in simultaneous oxidation of NADH. The change in the absorbance is directly proportional to the enzymatic activity, which is read colorimetrically. REAGENTS: 1. Phosphate buffer (pH 7.5) : Mixed 840ml of M/15 disodium hydrogen phosphate (11.876g

Na2 HPO4.2H2O/L) and 160 ml of M/15 disodium hydrogen phosphate (9.078g K2 HPO4/L) 2. Substrate- Alanine transaminase : Dissolved 5.0g of alanine and 20mg of α- oxoglutaric

acid in 20-30 ml of phosphate buffer and added 10%NaOH (0.5ml) to bring the pH to 7.5. Made up to 100ml with phosphate buffer. Kept in refrigerator.

3. Alanine citrate reagent : Dissolved 50g of citric acid in 50ml of distilled water and to this equal volume of redistilled alanine was added.

4. Dinitrophenyl hydrazine reagent - Dissolved 200mg of 2,4- Dinitrophenyl hydrazine in 85 ml of concentrated HCl and made upto a litre with water.

5. 0.4 N Sodium hydroxide. 6. Pyruvic acid : 100mg of pyruvic acid was dissolved in 100ml water. 7. Working standard : 1ml of pyruvic acid was made upto 100ml with distilled water. PROCEDURE: Pipetted 1ml of substrate in two tubes and placed in water bath at 37°C for a few minutes to reach this temperature. To one, the test 0.2 ml of serum was added and shaken gently to mix. Exactly 30 minutes later with the tubes still in the bath added 0.07ml alanine citrate reagent and 0.2ml serum to the other and the blank. The tubes were left for 20minutes and 1ml of DNPH reagent was added and left for another 20minutes and then added 10ml of 0.4N NaOH and read at 520nm using a green filter 10 minutes. Pipetted out various aliquots 3.5-7.5ml and made up the volume to 10ml with water. Added 1ml DNPH and 10ml of 0.4N NaOH as for the tests and the readings were taken at 520nm.

APPENDIX - 40 ESTIMATION OF ASPARTATE TRANSAMINASE (SGOT) (Reitman and Frankel, 1957) PRINCIPLE: SGOT catalyses the transfer of α- amino group from aspartate and to α- ketoglutarate with the formation of oxaloacetate and glutamate. Aspartate + α- ketoglutarate

SGOT Oxaloacetate + Glutamate.

Oxaloacetate + NADH+H

+ Malate + NAD

+

It catalyses the reduction of oxaloacetate to malate in simultaneous oxidation . REAGENTS: 1. Phosphate buffer (pH 7.5) : Mixed 840ml of M/15 disodium hydrogen phosphate (11.876g

Na2 HPO4.2H2O/L ) and 160 ml of M/15 disodium hydrogen phosphate (9.078g K2 HPO4/L). 2. Substrate- Aspartate transaminase : Dissolved 0.30g L- Aspartic acid and 15mg of

α- oxaloglutaric acid in 20-30ml of phosphate buffer. Kept in refrigerator. 3. Alanine citrate reagent : Dissolved 50g of citric acid in 50ml of distilled water and to this

equal volume of redistilled alanine was added. 4. Dinitrophenyl hydrazine reagent : Dissolved 200mg of 2,4- Dinitrophenyl hydrazine in 85 ml

of concentrated HCl and made up to a litre with water. 5. 0.4 N Sodium hydroxide. 6. Pyruvic acid- 100mg of pyruvic acid was dissolved in 100ml water. 7. Working standard : 1ml of pyruvic acid was made upto 100ml with distilled water.



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PROCEDURE: Pipetted 1ml of substrate in two tubes and placed in water bath at 37°C for a few minutes to reach this temperature. To one, the test 0.2 ml of serum was added and shaken gently to mix. Exactly one hour later with the tubes still in the bath added 0.07ml alanine citrate reagent and 0.2ml serum to the other and the blank. The tubes were left for 20minutes and 1ml of DNPH reagent was added and left for another 20minutes and then added 10ml of 0.4N NaOH and read at 520nm using a green filter 10 minutes. Pipetted out various aliquots 3.5-7.5ml and made up the volume to 10ml with water. Added 1ml DNPH and 10ml of 0.4N NaOH as for the tests and the readings were taken at 520nm.

APPENDIX - 41 ESTIMATION OF ALKALINE PHOSPHATASE

King and amstrong method (Varley et al., 1991) PRINCIPLE: The method used is that King and Armstrong in which Disodium Phenyl Phospate is hydrolysed with the liberation of phenol and disodium phosphate. The amount of inorganic phosphate so formed is estimated colorimetrically. REAGENTS: 1. Buffer substrate: 2g of Disodium phenyl phosphate per litre of Alkaline buffer, Filtered and

checked the pH. 2. Alkaline buffer pH 10 + 0.5: Dissolved 1.5g of Anhydrous sodium carbonate in about 500ml of water and then added

0.84g of sodium bicarbonate. Shook well and made up to a litre. Filtered if necessary and checked the pH.

3. 20% Trichloroacetic acid. 4. Acid Molybdate solution: Dissolved 5g of Ammonium Molybdate in 100ml of 5N sulphuric acid. (14ml of sulphuric acid

to 86ml of water). 5. Aminonaphthol sulphuric acid reagent. 6. Stock Phosphorus: Dissolved 35.1mg of Monopotassium Dihydrogen phosphate in 1.0ml of 10N sulphuric acid

and made up to 100ml with water. 7. Working standard: 10.0ml of stock standard solution was diluted to 100ml with water 1.0ml of the solution

contains 8 µg of phosphorus. PROCEDURE: Measured 6.0ml of buffer substrate into a test tube and placed in a waterbath at 37°C for a few minutes. Added 0.3ml of serum and stoppered the tube. Mixed and incubated for 15minutes at 37°C. Removed the tubes and added 1.2ml of 20% Trichloro acetic acid. Shook well and filtered. At the same time set up a control and blank. For the blank used 0.3ml of water and 6.0ml of buffer substrate. For the control added 0.3ml of serum and 6.0ml of water, to each tube added 1.2ml of 20% Trichloro acetic acid. Mixed and centrifuged. Pipetted out 5.0ml of each of the supernatant. Prepared a phosphate standard ranging from 1.5 and made upto 5.0ml with water. To all the tubes added 0.8ml of acid molybdate followed by 0.2ml of Aminonapthol sulphonic acid reagent. Mixed and allowed to stand for 20 minutes for the colour to develop. Read the colour developed using a red filter.

APPENDIX - 42 ESTIMATION OF ACID PHOSPHATASE

(Moog, 1946) PRINCIPLE: The enzyme phosphatase hydrolyzes p-nitrophenol phospate. The released p-nitrophenol is yellow in colour in alkaline medium and is measured at 405nm. The optimum pH for acid phosphatase is 5.3.



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REAGENTS: 1. Sodium hydroxide 0.085 N: Dissolved 0.85g sodium hydroxide in 250ml water. 2. Substrate solution: Dissolved 1.49g of EDTA, 0.84g citric acid and 0.03g p-nitrophenol phospate in 100ml water and adjust to pH 5.3. 3. Standard solution: Weighed 69.75mg p-nitrophenol and dissolve in 5.0ml distilled water (100mM). Enzyme extract: Homogenized 1g fresh tissue in 10ml of ice cold 50mM citrate buffer (pH 5.3) in a pre-chilled pestle and mortar. Filter through four layers of cheese cloth. Centrifuge the filtrate at 10,000g for 10min. Use the supernatant as enzyme source. PROCEDURE: 1. Incubated 3ml of substrate solution at 37°C for 5min. 2. Add 0.5ml enzyme extract and mix well. 3. Removed immediately 0.05ml and mix it with 9.5ml of sodium hydroxide 0.085 N. this corresponds to zero time assay. 4. Incubated the remaining solution (substrate + enzyme) for 5 min at 37°C. 5. Took 0.5 ml sample and mixed it with 9.5ml of sodium hydroxide solution. 6. Measure the absorbance of blank and incubated tubes at 405nm. 7. Take 0.2 to 1.0ml (4 to 20mM) of the standard, dilute 10.0ml with sodium hydroxide solution. Read the colour and the standard curve was drawn. CALCULATION: Specific activity is expressed as mM p-nitrophenol released per minute per mg.

APPENDIX 43 ESTIMATION OF LACTATE DEHYDROGENASE King’s Colorimetric Method (Varley et al., 1991)

PRINCIPLE: It is based on the formation of pyruvate dinitrophenylhydrazine. It uses lactate and measures the increase in colour as pyruvate is formed. REAGENTS: 1. Glycine buffer: Mixed 100m mol/L- 7.505g glycine and 5.85g sodium chloride/L in water. 2. Buffer substrate, pH 10.0: Added 125ml glycine buffer and 75ml 100m mol/L sodium

hydroxide to 5ml sodium lactate solution (BDX approx. 70%. Alternatively 4g lithium lactate may be used. This is more stable than the sodium salt.

3. NAD+ solution : 10mg/2ml in water and kept 4°C. NAD

+ is stabe if dissolved in Nicotinamide

solution (200 m mol/L.2.24 mg/ml). It is then kept up to 6 months at 4°C 4. 2,4-Dinitrophenyl hydrazine reagent : Dissolved 200mg of 2,4- Dinitrophenyl hydrazine in

h1mol/L HCl and made up to a litre with water. 5. Sodium hydroxide, 400m mol/L (16g/L) 6. Standard pyruvate solution, 1µmol/ml-11mg sodium pyruvate in 100ml buffered substrate. 7. NADH solution, 1µmol in buffer substrate (8.5mg/10ml assuring a purity of 85% ) usually

supplied as the disodium salt, mol.wt. 710 of purity from which the amount required can be calculated.

PROCEDURE: Pipetted 1ml of buffered substrate and 0.1ml serum diluted 1 to 5 with water into each of two tubes. Added 0.2ml of distilled water to one (the blank) and placed both tubes in a water bath at 37°C. Allowed to reach the temperature of the bath. Then to the other tube (the test) added 0.2ml NAD solution and shook to mix. Exactly 15 minutes after 1ml of DNPH reagent was added to each, shook to mix and kept in the water bath for further 15 minutes. Then removed



228

from the bath and to each added 10ml of 0.4N NaOH and read at 440nm within 1 to 5 minutes of addition of hydroxide.

APPENDIX - 44 ESTIMATION OF UREA

(Diacetyl Monoxime Method) (NIN Manual, 2003) PRINCIPLE: Serum urea reacts with acidic diacetyl monoxime during a short heating period. The presence of thiosemicarbazide and cadmium ions in the reagent intensifies the pink colour and decreases its photosensitivity, the colour is measured at 540nm. REAGENTS: 1. Colour reagent: Added 44ml of concentrated sulphuric acid and 66m ofl orthophosphoric

acid to about 100ml water in a 1 liter volumetric flask. Cooled the solution and added, 50mg thiosemicarbazide, 2.0 g of cadmium sulfate octahydrate and 10.0 ml of urea solution containing 0.26 mg urea (0.2 ml of standard containg 0.26ml of urea nitrogen/100ml). Mixed and diluted to the mark with water and transferred to amber bottle. This was stable for 6 months when refrigerated.

2. 2% Diacetyl Monoxime: Added 20g diacetyl monoxime to about 900ml water in 1 liter volumetric flask . Mixed to dissolve and diluted to the mark. Transferred to an amber bottle in which it was stable for at least 6 months when refrigerated.

3. Urea Nitrogen standard: Prepared a standard containing 60mg urea nitrogen/100ml by dissolving 128 mg urea in water with six drops of chloroform as a preservative. Diluted this stock 1:10 and preserved. Took 0.05, 0.01 and 0.15 ml of standard to obtain 15, 30, and 45mg urea nitrogen /100ml plasma, respectively.

PROCEDURE: To each of five test tubes labelled blank, standard 3.0, standard 6.0, standard 9.0 and unknown, added 5.0ml urea colour reagent. Transferred appropriate volume of urea nitrogen standard and 20µl serum, to their respective tubes and mixed. Added 0.5ml diacetyl monoxime reagent to all test tubes and mixed. Placed all the tubes in a boiling water bath for exactly 12 min. Removed the tubes, cooled in tap water and mixed. Read the absorbance of standards and unknown at 540nm against the reagent blank. CALCULATION: Urea nitrogen x 2.14 = Urea.

. APPENDIX- 45

ESTIMATION OF URIC ACID Caraway method (Varley et al., 1991)

PRINCIPLE: Uric acid reduces sodium phosphotungstate in alkaline solution to give a blue colour, which is measured colorimetrically. REAGENTS: 1. 14% Sodium carbonate solution. 2. Stock Folin uric acid reagent: Dissolved 50g of sodium tungstate in 400ml of distilled water. Added 40ml if 85% phosphoric

acid and refluxed gently for 2 hours. Cooled, transferred to a 500ml standard flask and made upto the mark with distilled water. The reagent was kept in brown bottle. The stock uric acid reagent was diluted one in ten before use.

3. Standard Uric acid: Weighed about 100mg of uric acid in a small beaker. Dissolved 60mg of Lithium carbonbate

in 15 to 20 ml of water in a test tube. Heated the solution to about 60°C and then added to the uric acid. Stirred well until it gets dissolved. Transferred it to 100ml standard flask. Added 2.0ml of 40% formaldehyde solution and then slowly with shaking added 1.0ml of 50% acetic



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acid as a preservative if it has to be preserved. Made up with water up to the mark and left it in the fridge.

4. Working standard: Diluted 2.0ml of the stock to 100 ml with water 1.0ml of this solution contains 20µg of uric

acid.

PROCEDURE: To 8.0ml of water, added 1.0ml of serum 0.5ml of 10% sodium tungstate and 0.5ml of 2/3 N sulphuric acid. Shook well and centrifuged after 15 minutes. 3.0ml of the supernatant was taken for the experiment. Took 0.5 to 2.5 ml of the working standard in a series of test tubes. Made up the volume to 3.0ml with water. To all the tubes added 1.0ml of the uric acid reagent and 1.0ml of 14% sodium carbonate solution and allowed to stand for 15 minutes. The blue colour developed was read in a colorimeter at 640nm.

APPENDIX- 46 ESTIMATION OF SERUM CREATININE

Alkaline picrate method (Varley et al., 1991) PRINCIPLE: Creatinine when treated with an alkaline picrate solution forms a red coloured complex. This is known as Jeffe’s reaction. The intensity of the colour developed is compared in the colorimeter at 540nm. REAGENTS: 1. 0.04M Picric acid. 2. 0.75N Sodium hydroxide 3. Stock standard solution: 100mg of creatinine was dissolved in N/10 Hydrochloric acid and made up to 100ml with the same. 4. Working standard: 2.0ml of the stock standard solution was diluted to 100ml with water. PROCEDURE: Took 3.0ml of water and added 2.0ml of serum and 1.0ml of 10% sodium tungstate solution and 2.0ml of 2/3N sulphric acid kept for 10 minutes and centrifuged 2.0ml of the supernatant was pipetted out in a test tube. Tubes containing 0.5-2.5ml of the working standard solution were taken and the volumes of these were made up to 3.0ml with water. Along with this a blank was also prepared. Added 1.0ml of 0.04M picric acid to all the tubes and 1.0ml of 0.75N Sodium hydroxide and left to stand for 20 minutes for the colour to develop. Shook well and the colour developed was compared with the standard against a reagent blank at 540nm. A graph was drawn taking the colorimeter readings on the Y axis and concentration of creatinine on the x axis. The concentration of creatinine in the test solutions were determined from the corresponding colorimerter readings.

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