CONTENTS

Sl.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12 13 14 15

Title of the Experiment

DETERMINATION OF SOLIDS IN WATER SAMPLE DETERMINATION OF RESIDUAL CHLORINE DETERMINATION OF HARDNESS OF WATER DETERMINATION OF DISSOLVED OXYGEN DETERMINATION OF BIOLOGICAL OXYGEN DEMAND DETERMINATION OF ACIDITY DETERMINATION OF ALKALINITY ESTIMATION OF CHEMICAL OXYGEN DEMAND DETERMINATION OF ORGANIC CARBON CONTENT IN SOILS DETERMINATION OF SLUDGE VOLUME INDEX DETERMINATION OF GROWTH CURVE USING E. coli ESTIMATION OF IRON USING PHENANTHROLINE METHOD ESTIMATION OF TURBIDITY OF WATER SAMPLE USING SPECTROPHOTOMETER SEPARATION OF CREAM FROM MILK ISOLATION OF SOIL MICROORGANISM USING SERIAL DILUTION METHOD

EXPERIMENT NO: 01

DETERMINATION OF SOLIDS IN WATER SAMPLE

AIM To determine the concentration of suspended solids, dissolved solids and total solids in a given water sample. APPARATUS REQUIRED Porcelain dish, measuring cylinder, weighing balance, beaker, filter paper, funnel. FORMULA Total solids in 20ml sample = (W2-W1) gm Concentration of total solids, x = (W2-W1) x 1000 mg/ml 20 Suspended solids in 20ml sample = (W4-W3) gm Concentration of suspended solids, y = W4-W3 x 1000 mg/ml 20 Concentration of dissolved solids = (x-y) mg/ml where, W1 = weight of dish (gm) W2 = weight of dish + dry residue (gm) W3 = weight of filter paper (gm) W4 = weight of filter paper + dry residue (gm)

PRINCIPLE

The total amount of solids present in water includes suspended solids and dissolved solids. Total solids in water sample can be determined by evaporating the sample and weighing the residue left behind. The suspended solids can be determined by filtering the sample with filter paper and measuring the weight of residue left in filter paper after drying. The difference between total solids and suspended solids will represent the dissolved solid content.

PROCEDURE

The empty dish was weighed and noted as W1 gm provided clean & dry. The given sample of 20ml was taken in the dish and evaporated. The dish was then again weighed with the residue left & noted as W2 gm. Now, the given filter paper was weighed as W3 gm. The given water sample was filtered through filter paper. This filter paper containing the residue is then dried and weighed as W4gm.

OBSERVATION

W1 = weight of dish

W2 = weight of dish + weight of dry residue = W3 = weight of filter paper W4 = weight of filter paper + dry residue CALCULATION = =

Total solid in 20 ml sample Concentration of total solid, x Suspended solids in 20ml sample

= (w2-w1)

= ((w2-w1)/20)1000 = = (w4-w3) = (x y) =

Concentration of suspended solids, y = ((w4-w3)/20)1000 = Concentration of dissolved solids RESULT i) ii) Concentration of total solids in the sample = =

Concentration of suspended solids in the sample =

iii) Concentration of dissolved solids in the sample =

EXP.NO:02

DETERMINATION OF RESIDUAL CHLORINE

AIM To determine residual chlorine in the given water sample. THEORY When filtered water is chlorinated by using chlorine or bleaching powder, the chlorine initially is consumed in killing bacteria, etc. and then in oxidizing the organic matter. When the break point is reached, then whatever chlorine is added to the water, it appears as residual chlorine. Hence for purified water, residual chlorine is tested by starch iodine test. When potassium iodine is added to the water sample, it will produce iodine ions in solution, which will react with chlorine to form iodine. Cl2 + 2I I2 + 2Cl-

In the presence of starch, the iodine produces a blue colour, which is taken as the evidence of residual chlorine. I2 + starch blue color

The intensity of the blue color produced can indicate the amount of chlorine residual. The total amount of chlorine residual present in the given water can also be quantitatively measured by titrating the iodine released with a standard solution of reducing agent, such as sodium thiosulphate. The end point of titration is indicated by the disappearance of blue colour. I2
+

2Na2S2O3 2NaI + Na2S4O6

PROCEDURE 1) 25 ml of the given water sample (2gm bleaching powder in 100 ml of water) was taken in a conical flask.

OBSERVATION

Qty of H2O S.No Sample (ml) 1 2 3 100 100 100

Test on H2O Sample Initial Value(ml) Final Value(ml)

Test on Distilled H2O Initial Value(ml) Final

Total amount of Residual

Value(ml) Chlorine(mg/l)

CALCULATION Residual Chlorine = (x-y)100035.5 mg/l 10040 where x = titre value of sample y = titre value of distilled water Residual Chlorine = mg/l

2) Small crystals of KI and distilled water were added to the above flask containing water sample to make 100 ml. 3) 0.5 ml of conc. HCI was added to reduce it to a low value between 3.5 to 4.2 to avoid the conversion of Cl into HOCI and OCl- . 4) The yellow coloured iodine solution was titrated against standard N/40 sodium thiosulphate solution till yellow colour becomes light. 5) 1ml of starch solution was added to the flask to produce blue colour. 6) Continue the titration with N/40 sodium thiosulphate till the blue colour disappears. 7) The total volume of titrate used in the entire titration was noted down. Let it be x ml 8) Distilled water was used in the place of water sample and entire procedure was repeated. The volume of titrate was noted as y ml. RESULT Amount of Residual Chlorine is mg/l EXP.NO:03

DETERMINATION OF HARDNESS OF WATER

AIM To determine the hardness of the given sample of water. REAGENTS HCl, (N/50) Na2CO3, EDTA, Methyl orange indicator, Phenolphthalein indicator, Erichrome Black-T indicator, MgSO4, Buffer solution. THEORY Hardness is caused by the presence of chlorides, sulphates and bicarbonates of calcium and magnesium. Hardness caused by calcium and magnesium bicarbonates is called temporary since it can be destroyed by boiling. Ca(HCO3)2 CaCO3 + H20 + CO2

Calcium Carbonate precipitates out and so hardness is removed. This form of hardness can be determined by simple titration with standard acid. Permanent hardness is caused by sulphates and chlorides of calcium and magnesium and is not affected by boiling. Total hardness is represented by titration with EDTA. Permanent hardness is determined as the difference between the total and temporary hardness. For the purpose of estimating total hardness, the most widely used is the disodium salt of EDTA. EDTA forms stable water soluble complexes with certain metal ions such as Ca and Mg. Erichrome Black-T solution is used as indicator and ammonium chloride solutions are added as buffer solutions to maintain the necessary pH.

OBSERVATIONS

Determination of Temporary Hardness Vol. of water sample 100 100 Burette Reading (ml) Initial Final Volume of HCl (ml), V1

S.No 1 2

Determination of Total Hardness Vol. of water S.No sample 1 100 2 100 Burette Reading (ml) Initial Final Volume of EDTA (ml), V3

Standardization of HCl

Vol. of water S.No sample 1 10 2 10

Burette Reading (ml) Initial Final

Volume of HCl (ml), V4

The equations for calculating hardness are: 1. Temporary hardness of water sample = (V2-V1) N2 50 1000 100 Where, N2 = Normality of HCL V1 = Vol. of HCl required for disappearance of pink color. V2 = Vol. of HCl required for changing yellow to orange red.

CALCULATIONS

1) Standardisation of HCl Normality of Na2C03 Volume of Na2CO3 = Normality of HCl Volume of HCl 0.01 10 = NHCl NHCl = N 2) Temporary hardness of water sample = (V2 V1) NHCl 50 1000 ppm 100 = ppm

3) Total hardness of water sample = V3 N4 50 1000 ppm 100 = ppm 4) Permanent hardness of water sample = Total hardness Temporary hardness = ppm

2. Total hardness of water sample = V3 N4 50 1000 100

where, N4 = Normality of EDTA V3 = Volume of EDTA 3. Permanent hardness of water sample = Total hardness Temporary hardness

PROCEDURE 1. Temporary Hardness Take 100ml of water under test into a clean conical flask after rinsing the flask with the same water. Add one or two drops of phenolphthalein indicator and titrate against approximately 0.01 N HCl. When the colour just disappears, note the titre value (V1, ml). Add methyl orange indicator and titration is continued till golden yellow just turns to orange red (V2, ml). 2. Total Hardness Measure out 100ml water under test into a conical flask. Add 3ml of buffer solution followed by 4 to 5 drops of Erichrome Black-T indicator. Mix thoroughly. The solution will become wine red in colour. It is then titrated against approximately 0.01 N EDTA. Contents of the flask are kept stirred throughout the titration. End point is the change of purple to blue colour persistent for about 15 seconds. Titre value is noted as V3 ml. 3 Standardization of HCl Na2CO3 can be used to standardize HCl. Prepare an approximately equal normal solution of Na2CO3 and titrate against HCl using methyl orange indicator.

RESULT i) ii) Temporary hardness of water sample = CaCO3 Permanent hardness of water sample = CaCO3

EXP.NO:04

DETERMINATION OF DISSOLVED OXYGEN

AIM To determine the amount of dissolved oxygen in a given water sample by Winklers method.

FORMULA USED Dissolved O2 (mg/l) = Volume of Na2S2O3 consumed x Normality of Na2S2O3 x 1000 x 8 Volume of water sample APPARATUS REQUIRED 300ml BOD bottle, conical flask, burette, measuring cylinder, pipettes.

REAGENTS REQUIRED 1. Std. Manganese sulphate solution (2M)

2. Azide Alkali iodide reagents (Na2N3 + NaOH +KI) 3. N/40 Sodium thiosulphate solution 4. Starch indicator (1g in 100 ml) 5. Conc. Sulphuric acid

THEORY When biodegradable organic matter is released into a body of water, micro-organism, especially bacteria, feed of the wastes breaking them down in to simple organic and inorganic substances. When these decompositions take place on an aerobic environment, stable end products such as carbondioxide, sulphates and nitrates are formed. Organic Matter + O2 CO2 + H2O + stable products (NO3 , PO4-)

When insufficient oxygen is available the result in anaerobic decomposition which is performed by completely different micro-organisms. They produce end products that can be highly objectionable including hydrogen sulphide (H2S), Ammonia (NH3) and methane. Organic Matter + O2 CO2 + H2O + stable product (H2S, NH3)

OBSERVATIONS For water sample, Vol. of water S.No sample

1 2 102 102

Burette Reading (ml) Initial Final

Volume of Na2S2O3 (ml)

For tap water, Vol. of tap water

102 102

S.No
1 2

Burette Reading (ml) Initial Final

Volume of Na2S2O3 (ml)

CALCULATIONS

For water sample, Dissolved oxygen = Volume of Na2S2O3 Normality of Na2S2O3 1000 8 Volume of water sample = mg/l For tap water, Dissolved oxygen = Volume of Na2S2O3 Normality of Na2S2O3 1000 8 Volume of water sample = mg/l

PROCEDURE 1. Take the sample in a BOD bottle. 2. Add 0.9 ml of conc. H2SO4 and 0.2ml KMnO4, then shake it for 5 min. 3. A yellow colour will appear. Then add 2ml MnSO4 + 3ml alkaline KI solution. If it doesnt show pH range below 8, then add some NaOH/KOH and mix it well. 4. If formation of any precipitate occurs, then add 1 ml conc.H2SO4 again. Then it is titrated against Na2S2O3 taken in burette using starch indicator. For titration take 102ml of the sample solution.

RESULT i) ii) The dissolved oxygen in the water sample is mg/l The dissolved oxygen in tap water is mg/l

EXP.NO:05

BIOCHEMICAL OXYGEN DEMAND

AIM To determine the biochemical oxygen demand (BOD) of the given sample. APPARATUS REQUIRED 1000ml of graduated cylinder, mixing rod, 300ml BOD bottles, pipette, BOD incubator, conical flask, burette. REAGENTS REQUIRED i. Std. Manganese sulphate solution (2M) ii. N/40 Sodium thiosulphate iii. Conc. H2SO4 acid iv. Starch Indicator v. Phosphate buffer solution THEORY BOD is defined as the amount of oxygen required by bacteria to stabilize decomposition of organic matter under aerobic condition. BOD is the major criteria used in stream pollution control, where organic loading is restricted to maintain desired dissolved oxygen level. It involves the determination of dissolved oxygen content of the sample before and after 7 days

of incubation at 200C. If the sample doesnt contain any oxygen, it is supplied with oxygen and depletion is calculated as BOD.

Dissolved O2(mg/L) = Vol.of Sodium thiosulphate consumedxConc.Of thiosulphatex1000x8 Vol. of water sample OBSERVATION

Initial DO Value Sl.no Volume of water sample(ml) 1 2 102 102 Burette reading Initial Final Volume of Na2S2O3(ml)

Final DO Value Sl.no Volume of water sample(ml) 1 2 102 102 Burette reading Initial Final Volume of Na2S2O3(ml)

CALCULATIONS Normality of Na2S2O3 used Initial Dissolved O2 (mg/l) = N

Volume of water sample taken for titration = ml = Volume of Na2S2O3 consumed x Normality x 1000 x 8 Volume of water sample = mg/l Final Dissolved O2 (mg/l) = Volume of Na2S2O3 consumed x Normality x 1000 x 8 Volume of water sample = mg/l BOD of given sample = Initial DO Final DO = mg/l PROCEDURE

1. The standard solution water taken into 1000ml graduated cylinder and half filled it without entrapment of air. 2. The pH of dilution water may range from 6.5 to 8.5. it is customary to buffer the solution by means of a phosphate system to about 7.0 3. Take the sample in BOD Bottle. Add 0.9 ml Conc.H2SO4 and and 0.2 ml KMnO 4 4. Take and shake it for 5 minutes. A yellow color appears. 5. Then add 2ml of MnSO4 and 3ml of alkaline KI solution. 6. If it didnt show yellow colour, then add some NaOH / KOH. 7. Then mix it. If there is any precipitate add 1ml of Conc. H2SO4 again. 8. Then it is titrated against Sodium thiosulphate using starch indicator.

RESULT i) ii) iii) Initial DO of the given sample = mg/l Final DO of the given sample = mg/l BOD of the given sample = mg/l

EXP.NO:06

DETERMINATION OF ACIDITY
AIM To determine the acidity of the given sample. PRINCIPLE Ions are present in the sample as a result of dissociation or hydrolysis of solutes heat with addition of standard alkali. Thus, acidity depends on the end point pH at indicator used. REAGENTS REQUIRED 1) Std. NaOH titration: Prepare 0.1N NaOH solution, standardise the solution by titrating

it with 40ml Potassium Hydrogen Thalate using 25ml burette. 2) CO2 free water: Prepare all stock and standardized solutions and dilution water with

distilled or de-ionised water that has been freshly boiled for 15mins and cooled at room temperature. Final pH of water should be greater than or equal to 6.0 and its conductivity should be less than 2 micromoles per cm. 3) Potassium Hydrogen Thalate: 15-20g of potassium hydrogen thalate is crushed to 100

mesh and dried at 1200C for 2 hours. It is cooled in a desiccator. After cooling weigh 100.5g.

Transfer it to a 1L volumetric flask and make it up to 1L. 4) 5) Standard NaOH (0.02N) Methyl orange indicator solution: Dissolve 50mg of methyl orange in distilled water

and dilute it to 100ml. 6) Phenolphthalein solution: 500mg of phenolphthalein is dissolved in 50ml of ethyl or

isopropyl alcohol. To this, add 50ml distilled water. Add NaOH solution dropwise until a faint pink colour appears. PROCEDURE 1) Methyl orange acidity or Mineral acidity: 25ml sample is taken in a 250ml conical flask (requiring not less than 25ml titrant). The sample is dechlorinised by adding 1 drop of 0.1N sodium thiosulphate solution for 1000ml. add 4 drops 0f methyl orange.

OBSERVATIONS Mineral Acidity Sl No. 1 2 Volume of sample (ml) 25 25 Burette reading (volume of NaOH)

Phenolphthalein Acidity Sl No. 1 2 Volume of sample (ml) 25 25 Burette reading (volume of NaOH)

CALCULATIONS Acidity in mg/L expressed as CaCO3 = Volume of NaOH Normality of NaOH 50 1000 Volume of Sample Mineral acidity (mg/L) = mg/L

Phenolphthalein acidity (mg/L)

= mg/L

Titrate it with 0.1N NaOH. The colour change is appearance of a faint orange colour. This titre value represents mineral acidity. 2) Phenolphthalein acidity or Total acidity at room temperature: To suitable aliquot of the

dechlorinated sample in a 250ml conical flask, add 4 drops of phenolphthalein indicator and titrate against NaOH till the appearance of a faint pink colour. While reporting result, the indicator used and temperature at which titration has been performed is also stated. INFERENCES Residual Cl2 interferes by bleaching colour of the indicator. It can be removed by the addition of sodium thiosulphate solution. A fading and impermanent end point characterizes the phenolphthalein acidity. RESULTS 1. The mineral acidity of the sample 2. The phenolphthalein acidity of the sample = mg/L = mg/L

EXP.NO:07

DETERMINATION OF ALKALINITY
AIM To determine the alkalinity of the given sample. PRINCIPLE Hydroxyl ions present in the sample as a result of dissociation or hydrolysis of solutes reacts with addition of standard acid. REAGENTS REQUIRED 1) Na2CO3 (1N) Weigh accurately 13 - 25 grams of anhydrous Na2CO3 (previously dried at 140 0C for 2 hours). Dissolve it in little distilled water and make up to 250 ml in a volumetric flask. 2) H2SO4 (N)

Add 28 ml of H2SO4 in 1000 ml volumetric flask and make up to 1 liter with CO2 free distilled water. Standardize it against 1N Na2CO3 solution using methyl orange as indicator. 3) Phenolphthalein indicator solution 500 mg of phenolphthalein is dissolved in 50ml of ethyl or isopropyl alcohol. To this, add 50ml distilled water. Add NaOH solution (0.02N) drop wise until a faint pink colour appears. 4) Mixed indicator solution Dissolve 20 mg of methyl red and 100 mg bromo cresol green in 100 ml of 95% ethyl alcohol or isopropyl alcohol (if Na salt of indicator is used, prepare the solution with distilled water instead of alcohol) OBSERVATIONS 1) Phenolphthalein alkalinity Sl No.

Volume of sample (ml)

Burette reading (ml; Volume of H2SO4)

1 2

2) Total alkalinity Sl No. Volume of sample (ml) Burette reading (ml;Volume of H2SO4) 1 2

CALCULATIONS Alkalinity in mg/L = Volume of H2SO4 Normality of H2SO4 50 1000 Volume of Sample Phenolphthalein alkalinity (mg/L) = mg/L

Total alkalinity (mg/L) = mg/L

PROCEDURE 1) Phenolphthalein alkalinity Place 25 ml of 50 ml of appropriate volume of sample in a conical flask (requiring not more than 25 ml titrate). Add 6 drops of phenolphthalein indicator solution. If no pink colour appears there is no phenolphthalein alkalinity, if appears, then titrate it with 1N H2SO4 until solution becomes colourless. 2) Total alkalinity Add 4 drops of mixed indicator to the sample in which Phenolphthalein alkalinity has been determined and titrate it against H2SO4. The colour change is from emerald green to pink. RESULTS 1. The phenolphthalein alkalinity of the sample = mg/L 2. The total alkalinity of the sample = mg/L

EXP.NO:08

ESTIMATION OF CHEMICAL OXYGEN DEMAND

AIM To determine the chemical oxygen demand in given sample by open reflux method.

APPARATUS AND CHEMICALS 1) Reflux Apparatus Consisting of 500 or 250 ml Erlenmeyer flasks with ground glass 24/40 neck and 300 mm 2) Standard Potassium Dichromate solution (0.25 N) 12.259 g of K2Cr2O7 , primary standard grade was dissolved in 1000ml distilled water at 1050C

3) Sulphuric Acid Reagent Con. H2SO4 containing 10g Ag2SO4/L was prepared. 4) Std. Ferrous ammonium sulphate Solution (0.25 N) 98g of Fe(NH4)2SO4.6H2O was dissolved in distilled water. 20ml of Con. H2SO4 was added and diluted to 1 litre 5) Ferroin Indicator 1.485 mg of 1,10 Phenanthroline monohydrate and 695 mg of FeSO4.7H2O was dissolved in 100ml of water.

THEORY Involved in determination of COD is that when the waste water sample in refluxed with a known excess of K2Cr2O7 in a 50% H2SO4 solution in presence of AgSO4 (as catalyst)

and HgSO4 (to eliminate interferenceof Cl) organic matter of sample in oxidized to H2O,CO2 and NH3. The excess dichromate remaining unreacted. The solution is titrated with standard solution of FAS.

PROCEDURE 25ml of sample taken in 500ml reflux, add 0.5g of HgSO4 and some porcelain pieces. Immerse the flask in cold water and slowly add 37.5ml of AgSO4 - HgSO4 reagent while continuously shaking flask. Add 12.5ml of 0.25N K2Cr2O7 solution, mix the content of the flask thoroughly. Attach the reflux condenser and reflux for 2 hours, wash the condenser with distilled water into the flask. Cool and distillate is diluted to about 150ml with distilled water. Add 2-3 drops of Ferroin indicator and titrate against 0.25N FAS. The color change at end point is from blue green to wine red. A blank titration is done by taking a volume of distilled water,equal to that of sample, into other flask. Add the same amount of reagents and reflux for 2 hours and titrate in the same way as sample titration. Record the volume of FAS used for titration as V2 ml. COD of sample corresponds to (V2- V1)ml of 0.25N FAS.

OBSERVATION

Sl No:

Volume of water sample (mL)

Burette Reading Initial 0 0 Final 2.3 4.2

Vol. of 0.25N FAS (mL) 2.3 4.2

1 2

Sample ( 12.5mL) Blank ( 12.5mL)

CALCULATIONS

Volume of 0.25N FAS used in the sample titration,V1 = 2.3 mL Volume of 0.25N FAS used in the blank titration,V2 Volume of sample used = 4.2 mL

= 25 mL = ((V2 V1) 0.25 8) / 25 = ((4.2-2.3)0.258)/25

COD of given sample of water

0.152 g/L

RESULT The COD of given sample of water by open reflux method = 0.152 g/L

EXP.NO:9

DETERMINATION OF ORGANIC CARBON CONTENT IN SOILS

AIM To determine the percentage of organic carbon present in soil. PRINCIPLE Organic Carbon is determined by sulphuric acid and aqueous potassium dichromate mixture. After the complete oxidation, the unused or residual dichromate is titrated against Ferrous Ammonium Sulphate (FAS). The used dichromate which is the difference between the original and the residual dichromate, gives a measure of the organic carbon content of the soil. 2K2Cr2O7 + 8H2SO4 TITRATION METHOD The reagents used are (a) K2Cr2O7 solution (1 M) Dissolve 49.024 grams of dry potassium dichromate in 800 ml of distilled water and dilute it to 1 litre 2K2SO4 + 2Cr2(SO4)3 + 6[O]

(b) (c)

Concentrated H2SO4 Ferrous Ammonium Sulphate (FAS) Dissolve 78.390 grams of FAS powder in 50 ml of concentrated

H2SO4 and dilute it to 1 litre with distilled water. Standardise it by adding ferroin indicator. FORMULA USED % Organic Carbon Content = (Titre value) 0.2 0.3 / (Sample weight)

OBSERVATIONS

Sl No.

Weight of Sample (g)

Burette Reading (ml)

Volume of FAS (ml)

Initial 1. 2.

Final

CALCULATIONS % Organic Carbon Content = (Titre value) 0.2 0.3 / (sample weight) = = %

PROCEDURE 1. Weigh 0.2 gram of ground soil. 2. Add 5 ml of K2Cr2O7 and 7.5 ml of Conc. H2SO4 to the sample. 3. Place the container in a pre-heated block at 1450C 1500C for exactly 30 minutes. 4. After 30 minutes, remove it and allow it to cool. 5. Transfer the digest to a 100 ml conical flask and add 0.3 ml of ferroin indicator. 6. Using the stirrer, titrate the flask using the FAS solution. The end point is the colour change from green to brown. RESULT The percentage of organic carbon content in the soil = %.

EXP.NO:10

DETERMINATION OF SLUDGE VOLUME INDEX

AIM To determine the sludge volume index (SVI) of a given water sample. APPARATUS REQUIRED Settling column use 1 liter graduated cylinder equipped with a stirring mechanism not more than 4 rpm. Stop watch, Thermometer, Filter paper, Hot Air oven. FORMULA REQUIRED Sludge volume index = Settled sludge volume x 1000 Suspended solids

Total Suspended (in mg/L) =

(A-B) x 1000 Sample Volume

Where, A = Weight of filter + dry residue (mg) B = Weight of filter (mg)

THEORY A calculation that indicates the tendency of activated sludge solids (aerated solids) to thicken or to become concentrated during the sedimentation/thickening process. By definition, the sludge volume index (SVI) is the volume in millilitres occupied by 1 g of a suspension after 30 min settling.

PROCEDURE Calculation of Settled sludge volume 1. 1 litre of sample was placed in settling volume and dissolved solids by covering the top. 2. Stirring rod was inserted and stirring mechanism was activated. CALCULATIONS = 6.510-2 m = 210-3 m = D2H 4 = (6.510-2)2210-3 4 = 6.64 ml/L

Diameter , Height ,

D H

Volume of settled sludge

Weight of filter paper + Dry residue, A Weight of filter paper (dry), B

g = g

Total suspended solids

= (A-B) x 1000 Sample Volume = = g/L

Sludge volume index

= Settled sludge volume x 1000 Suspended solids = (ml/L) (g/L)

= ml/g

3. The stop watch was started and the suspension was allowed to settle. 4. There was a continuous stirring throughout the test. 5. Suspension temperature driving test was maintained at that in the basis from which sample was taken. 6. The volume occupied by suspension was determined after 30 min (V= r2h )

For Total Suspended solids 1. A sample volume of 50ml is taken 2. Its allowed to pass through a filter paper. 3. Filter paper was rinsed to avoid base reactions. 4. Filter paper is dried for 1 hr in oven. 5. Cooled and weight is taken.

RESULT Sludge Volume Index (SVI) of the given sample = ml/g

EXP.NO:

DETERMINATION OF GROWTH CURVE USING E. coli

AIM To determine the growth curve studies using yeast and E. coli.

APPARATUS REQUIRED Autoclave, Laminar chamber, Weighing machine

CHEMICALS REQUIRED Peptone NaCl Beef Extract Yeast Extract Malt Extract pH = = = = = = 5 g/lit 5 g/lit 1.5 g/lit 1.5 g/lit 1.0 g/lit 7.4 0.2

AUTOCLAVE CONDITIONS Pressure Temperature = = 15 psi 120 0 C

Pressure release time =

30 min

THEORY

Batch culture is a closed culture system which contains an initial limited amount of nutrient. The inoculated culture will pass through a number of phases. After inoculation, there is a period during which it appears that no growth takes place. This period is referred to as lag phase and may be considered as time of adaptation. In the commercial phase, length of lag phase should be reduced as much as possible and this may be achieved by using suitable inoculums. Following a period during which the growth rate of the cell gradually increases. OBSERVATIONS

Culture (min of incubation) 0 30 60 90 120 180 240 300 360 1380 1440

Absorbance OD(600nm)

Table 1.1: Data for adsorbance and time of incubation

The cells grow under constant maximum rate and this period is known as log or exponential phase. The population is most nearly uniform in terms of chemical composition of cells, metabolic activity and other physiological characteristics. They tend toward cessation of growth can be attributed to a variety of substances. Particularly the exhaustion of some nutrients and production of toxic products during growth. The population remains constant for the time since the reproduction rate is balanced by an equivalent death rate. This is referred to as stationary phase. During the death phase, number of viable cells decreases exponentially. Essentially, the inverse of growth during log phase. A variety of condition contributes to cell death but the most important are depletion of essential nutrients and accumulation of inhibitory products such as acid. Six characteristic phases of the growth cycle are recognized. 1. Lag Phase: Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. 2. Acceleration Phase: Once after the cells have adapted to the new environment, cell division occurs at increasing frequency until maximum growth rate reached.

3. Exponential (log) Phase: The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time (below) derive.

Bacteria growth rate vs time

4. Deceleration Phase: When level of substrate decreases, it eventually become limiting and no longer sustain maximum growth rate. 5. Stationary Phase: Exponential growth cannot be continued forever in a batch culture. Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space". During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle. It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process. 6. Death Phase: If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase. PROCEDURE

1. Prepare the nutrient broth and sterilize it in the autoclave. 2. After sterilizing aseptically transfer 5ml of broth culture to 15ml of nutrient broth.

3. Determine the initial OD at 600nm. 4. Place the inoculated culture flask in the shaker at 37C for 16 hr for incubation. 5. Take 5ml of culture from the incubated flask and inoculate it into 100ml of sterile nutrient broth aseptically. 6. Determine the OD of the sample at 600nm. 7. Repeat the step 6 at each 30 min interval for a period of 8hrs.

A: Acceleration Phase Figure : Bacteria growth curve B: Deceleration Phase

RESULT

The absorbance for different time intervals is noted, growth curve is plotted and four distinct phases of growth curve are identified from the graph.

ESTIMATION OF IRON USING PHENANTHROLIN METHOD

AIM To determine the amount of iron present in the given sample of water using phenanthrolin method. PHENANTHROLIN METHOD PRINCIPLE Iron is brought into solution, reduced to ferrous state by boiling with acid and hydroxyl amine, and treated with 1,10-phenanthrolin at a pH of 3.2-3.3. This forms an orange-red complex (3 molecules of phenanthrolin thalate each molecule of iron to form the orange-red complex). The coloured solution follows Beers Law; its intensity is independent of pH from 3 to 9. INTERFERENCE Strong oxidising agents like cyanide, nitrates, phosphates (poly phosphates other than anthrophos), chromium, zinc, in concentrations 10 times that of Fe, Co and Cu, in excess of 5mg/l and Na in excess of 2mg/l causes interference. Bi, Cd, Hg, Mo and Ag precipitates phenanthrolin. Adding excess hydroxyl amine eliminates errors caused by excessive concentration of strong oxidising agents in the presence of interfering metal ions; use a large excess of phenanthrolin to replace that complexed by the interfering metals. REAGENTS Use reagents low in Fe. Store reagents in glass stoppered bottles. HCl and Ammonium Acetate buffer solution are stable indefinitely of tightly stoppered. Hydroxyl amine, phenanthrolin, and stock iron are stable for several months. The standard iron solutions are not stable.

Absorbance v/s Concentration Plot

0.35 0.3 0.25 Absorbance 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 Concentration (mg/ml)

SCALE x axis, 2cm = 5mg/ml y axis, 2cm = 0.05

1. HCl. 2. Ammonium Acetate buffer solution :- Dissolve 250g of ammonium acetate in 150ml of water. Add 700ml bleaching acetic acid. 3. Hydroxyl amine solution :- Dissolve 10g hydroxyl amine in 100ml water. 4. Phenanthrolin solution :- Dissolve 100mg 1,10-phenanthrolin mono hydrate in 100ml water by stirring and heating to 800C. Do not boil. As it dissolves the solution darkens. Heating is not necessary if two drops of conc. HCl is added to the water. 5. KMnO4 (0.1 N) :- Dissolve 0.311g of KMnO4 in distilled water and dilute it to 100ml. 6. Stock and iron solution :- Use a) Metal (electrolytes iron wire) and; b) Salt for preparing (FAS) Slowly add 20ml of conc. H2SO4 to 50ml of water and dissolve 1.404g of FAS. Add 0.1m KMnO4 drop wise until a faint pink colour presents. Dilute to 1000ml with water. 7. Standard iron solution :- Prepare 50ml stock solution into 1000ml volumetric flask and dilute it to mark with water 1ml of this solution is equivalent 10 Fe. PROCEDURE Mix the sample thoroughly and measure 50ml into a conical flask. Add 2ml conc. HCl and 1ml hydroxyl amine solution. Add few glass beads and heat to boiling. Continue boiling until the volume is reduced to 15 or 20ml. Cool to room temperature and transfer it to a 50ml volumetric flask. Add 10ml of Ammonium Acetate buffer solution and 4ml phenanthrolin solution and dilute it to mark with distilled water. Mix thoroughly and allow a minimum of 10 minutes for maximum colour development. Prepare a series of standards. Measure the absorbance at 510nm, using cuvetts. Use distilled water as reference. RESULT The mg/ml Fe in sample were calculated as mg/ml.

OBSERVATION Determination of Total Solids Wt. of empty S.No Dish(W1) gm 1 Wt of Dish + Total solids in 20ml of Concentration of total Wt of residue Sample (W2 W1) gm Solids = W2 W1 x 1000 (W2) gm 20 (mg/ml)

Determination of Suspended Solids Wt. of filter S.No paper (W3) gm Wt of filter paper + dry residue (W4) gm Suspended solids in 20ml of Sample (W4 W3) gm Conc of suspended Solids = W4 W3 x 1000 20 (mg/ml)

Concentration of dissolved solids = Conc. of total solids Conc. of suspended solids = = mg/ml