LAB MANUAL

School of Sciences, P P Savani University

Cleaner Production & Waste Utilization Practical (SSES3230)

Semester V, B.Sc. Environmental Science

NH No.: 8, Village: Dhamdod, Ta. Mangrol, Near Kosamba, Surat – 394 125. (GUJARAT).

LAB MANUAL

INSTITUTE NAME : SCHOOL OF SCIENCES

DISCIPLINE : ENVIRONMENTAL SCIENCE

COURSE : B.SC. (H.)

SUBJECT CODE : SSES3230

SUBJECT NAME : Cleaner Production & Waste Utilization Practical

 NH No.: 8, Village: Dhamdod, Ta. Mangrol, Near Kosamba, Surat – 394 125. (GUJARAT).

CERTIFICATE
Date:

INSTITUTE NAME SCHOOL OF SCIENCES COURSE

DISCIPLINE ENROLLMENT ID

STUDENT NAME

ACADEMIC YEAR SEMESTER

SUBJECT CODE

SUBJECT NAME

PERFORMED
TOTAL PRACTICAL
PRACTICAL

SUBJECT CONVENOR EXTERNAL EXAMINER

PRINCIPAL INSTITUTE SEAL

LIST OF PRACTICALS:

S. Name of Practical Page Date

No. No.
1 Working of instruments in Environment Audit Laboratory 2
2 Determination of all Physical Parameters of a given sample 7
3 Determination of all Chemical Parameters of a given sample 11
4 Determination of biological Parameters of a given sample 21
5 Determination of microbiological Parameters of a given sample 23
6 Testing a soil sample for all its parameters 27
7 Case studies on successful implementation of cleaner 31
production
8 Case studies on waste utilization from specific industries 32

P a g e 1 | 30
Practical: 1. Working of Instruments in Environment Audit Laboratory

1. pH meter:
It is electric device used to measure hydrogen-ion activity
(acidity or alkalinity) in solution. Fundamentally,
a pH meter consists of a voltmeter attached to a pH-responsive
electrode and a reference (unvarying) electrode. The pH-
responsive electrode is usually glass, and the reference is
usually a mercury–mercurous chloride (calomel) electrode,
although a silver–silver chloride electrode is sometimes used.
When the two electrodes are immersed in a solution, they act as
a battery. The glass electrode develops an electric
potential (charge) that is directly related to the hydrogen-ion
activity in the solution (59.2 millivolts per pH unit at 25 °C [77
°F]), and the voltmeter measures the potential difference
between the glass and reference electrodes.

2. BOD Incubator:
BOD incubator is the most versatile and reliable low
temperature incubator which is designed to maintain at 20°C,
necessary for Biological Oxygen Demand/Biochemical Oxygen
Demand (BOD) determination. BOD incubators provide
controlled temperature conditions for accelerated tests and
exposures.

3. TDS/ Conductivity Meter:

TDS meter is an equipment which is used to measure the
amount of Total Dissolved Solids i.e. TDS in an aqueous
state/water. The instrument/equipment is convenient,
accurate, portable and easy to operate, especially for field
measurement. A TDS meter is based on the Electrical
Conductivity (EC) of water. Pure H20 has usually zero
conductivity. Conductivity is usually about 100 times the
total cations or anions expressed as equivalents. TDS is
calculated by converting the EC by a factor of 0.5 to 1.0 times
the EC, depending upon the levels. Typically, the higher the
level of EC, the higher the conversion factor to determine
the TDS. While a TDS meter is based on conductivity, TDS
and conductivity are not the same thing.
P a g e 2 | 30
4. Weigh Balance:
It is a class of balance designed to measure small mass in the
sub-milligram range. The measuring pan of an analytical
balance (0.1 mg or better) is inside a transparent enclosure
with doors so that dust does not collect and so any air currents
in the room do not affect the balance's operation.

5. Muffle Furnace:
A muffle furnace, is an oven type equipment that can reach high
temperatures. The use of a muffle furnace is to burn off specific
types of compounds. For instance, for a standard determination
of the amount of inorganic components within an object, a
muffle furnace can be used to burn off all the organic material
(which is roughly said always < 500°C). It objective thereof is
plainly to auto-ignite the organic material, due to high
temperatures.

6. COD Digester:
COD digester will be used for potassium dichromate -
sulphuric acid digestion of water/waste water samples.

7. Magnetic stirrer with hot plate:

It is used to cause a stir bar immersed in a liquid to spin very
quickly, agitating or mixing the liquid. A magnetic stirrer often
includes a provision for heating the liquid.

8. Turbidity meter:
Turbidity meter is used to measure turbidity using
photometric techniques is defined as reduction of
transparency of a liquid caused by the presence of
undissolved matter. Reduction of transparency can be
measured to a certain extent using scattering and/or
transmission of light.

9. Flame photometer:
The objective of flame photometer is based on the
measurement of the emitted light intensity when a metal is
introduced into the flame. The wavelength of the colour gives
information about the element and the colour of the
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 flame gives information about the amount of the element
present in the sample.

10. DO Meter:
DO meter is used to measure the dissolved oxygen present in the
water samples

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 Practical: 2 Determination of all the Physical Characteristics of the wastewater
sample
1. Temperature

Objective: To determine the temperature of given water sample.

Apparatus: Thermometer and beaker etc.

Method:

Take the water sample in three different beakers and dips the thermometer in the first
beaker for 2 minutes and notes the temperature in the record file. This process is repeated
with second and third beaker and notes the temperature reading in the record. Calculate the
average temperature with this data.

Sr. Type of Sample Temperature °C

No.

2. Turbidity

Objective: To determine the turbidity of given water sample.

Apparatus: Nephelo turbidity meter with sample cells, volumetric flask and amber glass
bottle etc.

Reagents:
(a) Solution I- Dissolve 1.0 gm hydrazine sulphate, (NH2)2, H2SO4 in distilled water and
dilute to 100 ml in a volumetric flask.
(b) Solution II- Dissolve 10 gm hexamethylenetetramine, (CH2)6N4, in distilled water and
dilute to 100 ml in a volumetric flask.
(c) 4000 NTU suspension- In a flask mix 5.0 ml of solution I and 5 ml of solution II. Let
stand for 24 hours at 25±3 0C. This result in a 4000 NTU suspension. Store in an
amber glass bottle. The suspension is stable for up to 1 year.

Page 7 of 33
 (d) Dilute 4000 NTU stock solution with distilled water to prepare dilute standards just
before use and discard after use.

Method:

Calibrate the Nephelo turbidity meter with the 100 NTU suspensions and verify the setting
with a reading on the 10 NTU suspensions. Then thoroughly shake sample, wait until air
bubbles disappear and pour sample into turbidity meter tube. Read turbidity directly from
instrument display. In case the turbidity is going beyond the scale dilute the sample and
measure the turbidity. The dilution factor should be taken into account for the final
calculation of the result.

Sr. Type of Sample Turbidity (NTU)

No.

3. Conductivity

Objective: To determine the Conductivity of Wastewater Samples

Apparatus: Conductivity meter, distilled water, beaker.

Method:
• Take Water Sample from different locations.
• Turn on the EC meter and calibrate the probe using a standard solution of known
conductivity. Check calibration by measuring the EC of the standard solutions in measure
rather than calibrate mode.
• Collect sample water in a glass or plastic container. Collect enough so the probe tip
can be submerged in sample; either rinse the probe with deionized water (and blot dry) or
with sample before inserting the probe into the collection vessel.
• Submerge the probe into the sample and wait until the EC reading on the meter
stabilizes. Many meters have automatic temperature correction (ATC), which calculates
the EC taking into account temperature, if your meter does not have this feature, you may
Page 8 of 33
 need to adjust a knob on the meter to correct the EC for temperature. Record the
measurement when the EC reading is stable.

OBSERVATION TABLE:

Sr. No Type of Sample Conductivity (µS)

4. pH
Objective: To determine the pH value of Wastewater Samples.

Apparatus: pH meter, distilled water, beaker.

Method:
• Take Water Samples from different locations.
• Turn on the pH meter and calibrate the probe using two standard solutions (pH 4, 7,
and 10 buffers are recommended, dependant on the range you are measuring).
• Check calibration by measuring the pH of the standard solutions in measure rather
than calibrate mode.
• Collect sample water in a glass or plastic container. Collect enough so the probe tip
can be submerged in sample; either rinse the probe with deionized water (and blot dry) or
with sample before inserting the probe into the collection vessel.
• Submerge the probe into the sample and wait until the pH reading on the meter
stabilizes. Many meters have automatic temperature correction (ATC), which calculates
the pH taking into account temperature, if your meter does not have this feature, you may
need to adjust a knob on the meter to correct the pH for temperature. Record the
measurement when the pH reading is stable.

OBSERVATION TABLE:

Sr. No Type of Sample pH Value

Page 9 of 33
➢ OVERALL RESULT:

➢ OVERALL CONCLUSION:

Page 10 of 33
Practical: 3 Determination of all the Chemical Characteristics of the wastewater
sample

1. Total solids

Objective: To determine the total solids in given water sample.

Apparatus: Porcelain dishes, Measuring cylinder, Weighing Balance and Hot Air Oven.

Method:

• Weight the previously dried empty porcelain dishes (W1) already kept at 105°C for
2 hours and in desiccator for 30 minutes.
• Take 100 ml well mixed sample in each porcelain dishes and place the porcelain
dishes in the oven at 105°C carefully for 24 hours.
• After 24 hours, take the dishes out and keep in the desiccator for 30 minutes for
cooling. Now weight the porcelain dishes and note the final weight (W2).
• Repeat the process of weighing and drying till constant weight is achieved.

OBSERVATION TABLE:

Sr. Type of Sample Initial Weight (mg) Final weight (mg) Total
No. Solids(mg/L

Calculation:

TS (mg/l) = (W2-W1) x 10^6 / Sample volume (ml)

W1=Initial Weight of the dish, mg

W2=Final Weight of the dish, mg

Amount of Sample taken= ml

Page 11 of 33
2. Total Suspended Solids (TSS)

Objective: To determine the TSS of given water sample.

Apparatus: Filter paper (Whatman no-41), beaker, hot plate, physical balance,
funnel, measuring cylinder, desiccator, oven etc.

Method:
• Dry the filter paper in oven (105°C) and cool in desiccator then weight it.
• Then filter 100 ml sample through filter paper.
• Keep the filter paper in oven for overnight to dry, cool the filter paper in a
desiccator and again weight it.
• Calculate the TSS using following formula

OBSERVATION TABLE:

Sr. Type of Sample Initial Weight of Final weight of Filter Total

No. Filter Paper (mg) Paper (mg) Suspended
Solids(mg/L)

Calculation:

W2-W1 x 1000/V
Where,
W1 = Initial weight of filter paper, mg
W2 = final weight of filter paper, mg
V = Volume of sample, ml

Amount of Sample taken= ml

Page 12 of 33
3. Total Dissolved Solids

Objective: To determines the TDS of given water sample.

Apparatus: Filter paper (Whatman no-41), Beaker, Hot plate, Physical balance,
Funnel, Measuring cylinder, Desiccator, Hot Air Oven

Method:
• Dry the beaker in oven (105°C) and cool in desiccator then weight it.
• Then Take 100 ml filtered sample in this beaker and evaporate up to dryness on a
hot plate, cool in a desiccator and again weight it.
• Calculate the TDS using following formula

OBSERVATION TABLE:

Sr. Type of Sample Initial Weight of Final weight Total

No. beaker (mg) of beaker (mg) Dissolved
Solids(mg/L

Calculation:

W2-W1 x 1000/V
Where,
W1 = Initial weight of beaker, mg
W2 = final weight of beaker, mg
V = Volume of sample, ml

Amount of Sample taken= ml

4. Alkalinity

Objective: To determine the alkalinity of given water sample.

Apparatus: Burettes, Pipette, Volumetric flask, conical flask and Beaker.

Page 13 of 33
 Reagents:
1. Standard sulphuric acid (0.02N) - Prepare the 0.1 N H2SO4 by diluting 30 ml conc.
H2SO4 to 1 litre distilled water. Standardized it with standard sodium carbonate
Na2CO3 0.1N. Dilute appropriate volume of sulphuric acid, approximately 100 ml to
500 ml to obtained standard 0.02N H2SO4.
2. Phenolphthalein indicator solution (alcoholic, pH 8.3) - Dissolve 5 gm
phenolphthalein in 500 ml 95% ethyl alcohol. Add 500 ml distilled water.
3. Methyl orange indicator- 0.05 gm methyl orange diluted 10 100 ml with distilled
water.

Method:
• Take 100 ml sample in volumetric flask, add 2-3 drops of Phenolphthalein
indicator.
• If pink colour develops, titrate with 0.02N H2SO4 till a colour disappears.
• Note the volume of sulphuric acid used. Add 2-3 drops of Methyl orange indicator
to the same flask and continue titration till yellow colour changes to orange.
• Note the volume of sulphuric acid used.

OBSERVATION TABLE:

Sr. Type of Sample H2SO4 used With H2SO4 used with

Phenolphthalein Methyl
No. indicator (ml) A Orange indicator (ml) B

Calculation:

Calculate total (T), Phenolphthalein (P) and Methyl orange (M) alkalinity as follows and
express in mg/l as CaCO3.

Phenolphthalein alkalinity (mg/l as CaCO3) = A x N x 1000/ volume of sample

Total alkalinity (mg/l as CaCO3) = B x N x 1000/ volume of sample
Methyl orange alkalinity (mg/l as CaCO3) = (B-A) x 1000/ volume of sample

Amount of Sample taken= ml

Page 14 of 33
 5. Acidity

Objective: To determine the acidity of given water sample.

Apparatus: Burettes, pipette, Volumetric flask, Conical flask and Beaker.

Reagents:

(a) Standard sodium hydroxide (0.02N) - 0.8 gm sodium hydroxide is dissolved in

distilled water and dilute to 1000 ml using carbon dioxide free distilled water.
Standardized against standard oxalic acid solution.
(b) Phenolphthalein indicator- 0.05 gm Phenolphthalein is dissolved in 500 ml 95%
ethyl alcohol 500 ml distilled water is added. Add drop wise 0.02N sodium
hydroxide till faint pink colour appears.

Method

• Take 100 ml sample in conical flask add 2 drops of Phenolphthalein indicator

and titrate with standard NaOH solution till pink colour appears.

OBSERVATION TABLE:

Sr. Type of Normality of NaOH used Acidity (mg/l as CaCO3)

NaOH (ml)
No. Sample

Calculation:

Acidity as CaCO3 (mg/l) = ml titrant used (NaOH) x 1000/ ml sample taken

Amount of Sample taken= ml

Page 15 of 33
6. Total Hardness

Objective: To determine the total hardness of given water sample.

Apparatus: Volumetric flask, Conical flask, Burette, Pipette, Measuring Cylinder,

Burette stand, Pipette stand and Wash bottle

Reagents-
(a) Buffer solution- Dissolve 16.9 gm NH4Cl in 143 ml conc. NH4OH. Add 1.25 gm
magnesium salt of ethylene diamine tetra acetic acid (EDTA) and dilute to 250 ml
with distilled water. Store in a plastic bottle stopper tightly for no longer than one
month.
(b) Eriochrome Black T solution (as indicator)- Dissolve 0.5 gm dye in 100 ml

triethanolamine or 2 ethylene glycol monomethyl ether. The salt can also be used in
dry powder form by grinding 0.5 gm dye with 100 gm NaCl.

(c) Standard EDTA titrant (0.01M)- Weight 3.723 gm di-sodium salt of EDTA,
dehydrate, dissolve in distilled water and dilute to 1000 ml. Store in polyethylene
bottle.
(d) Standard calcium solution- Weight 1 gm anhydrous CaCO3 in a 500 ml flask. Add
1 + 1 HCl slowly through a funnel till all CaCO3 is dissolved. Add 200 ml distilled
water and boil for a few minutes to expel CO2. Cool and add a few drops of methyl
red indicator and adjusts to the intermediate orange colour by adding 3N NH4OH or
1 + 1 HCl, as required. Transfer quantitatively and dilute to 1000 ml with distilled
water, 1 ml=1mg CaCO3.

Method:
• Standardize the EDTA titrant against standard calcium solution using procedure
given below
• Take 50 ml sample in a conical flask add 2 ml buffer solution and 2 drops EBT
indicator solution, wine red colour appears.
• Titrate with EDTA till the colour changes to blue. Note the volume of EDTA used.
• Calculate the total hardness by following formula-

OBSERVATION TABLE:

Sr. Type of EDTA Total Hardness(mg/L as

used CaCO3)
No. Sample (ml)

Page 16 of 33
 Calculation:
Total Hardness (EDTA) mg/L= A x 1000/ volume of sample
Where,

A= ml EDTA used

Amount of Sample taken= ml

7. Chemical Oxygen Demand (COD) (Open Reflux Method)

Objective: To determine the COD of given water sample.

Apparatus: COD reflux assembly, Conical flask, Beaker, Measuring cylinder, Wash
bottle, Burette stand, Pipette stand, Burette, Pipette.

Reagents-
(i) Standard potassium dichromate solution (0.25N) - 12.259 gm K2Cr2O7 is
dissolved, previously dried at 1030C for 24 hours, in 1000 ml distilled water.
(ii) Sulphuric acid reagents (0.1N)- Add 22 gm silver sulphate (AgSO4) to a 9 lbs (4
kg) bottle of concentrated H2SO4. Keep overnight for dissolution and after that
shake well to it.
(iii) Standard ferrous ammonium sulphate solution (0.1N) - Dissolve 39.2 gm of Fe
(NH4)2 (SO4)2.6H2O in 500 ml of distilled water. Add 20 ml of concentrated H2SO4,
and dilute to 1 litre.
Standardization- Dilute 10 ml standard K2Cr2O7 solution to about 100 ml. Add 30
ml conc. H2SO4 and cool. Add 3-4 drops of ferroin indicator and titrate with the
ferrous ammonium sulphate solution till the colour changes to wine red. The
normality of the Fe (NH4)2 (SO4)2 solution is given by the following formula-
(iv) Ferroin indicator solution- 1.485 gm. 1-10 phenanthroline monohydrate and
695 mg ferrous sulphate FeSO4.7H2O are added in distilled water and dilute to
1000 ml.
(v) Mercury sulphate (HgSO4) - Crystal or powder.

Method-
• 20 ml of sample is taken into a refluxing tubes. 1 gm HgSO4 and very slowly
added 30 ml sulphuric acid reagent with mixing to dissolve HgSO4 cool, while
mixing to avoid possible loss of volatile materials.
• 10 ml K2Cr2O7 solution is added and mix.
• Tubes are placed in COD Digester.
• Reflux for 2 hours, cool, and wash down condenser with distilled water.

Page 17 of 33
 • Cool to room temperature and remaining 80 ml Distilled Water is added
through open end of condenser, once the digestion is completed.
• Titrate it with F.A.S. using 0.1-0.15 ml (2-3 drops) ferroin indicator.

OBSERVATION TABLE:

Sr. Type of F.A.S. used (ml) COD (mg/l)

No. Sample

Calculation:

To calculate normality of FAS,

N1 * V1= N2* V2
Where,
N1=Normality of FAS, N
V1= Volume of FAS used in titration, ml
N2=Normality of K2Cr2O7, i.e. 0.25 N
V2= Volume of K2Cr3O7 used i.e. 10 ml

To Calculate COD,

COD (mg/L) = [(B-A)*8000*N1]/ml of Sample

Where,
B= ml of FAS used in Blank
A= ml of FAS used in Sample
Amount of Sample taken= ml

Page 18 of 33
8. Dissolved Oxygen D.O. (Modified Winkler’s method)

Objective: To determine the concentration of dissolved oxygen in given water

sample.

Apparatus: BOD bottle, Pipette, Burette, Conical flask, volumetric flask, Beaker,
Measuring Cylinder, Burette stand, Burette and Pipette.

Reagents:
(i) Sodium thiosulphate [Na2S2O3.5H2O] (0.025 N) - dissolve 6.205 gm of solution
thiosulphate in previously boiled distilled water add 0.4 gm solid NaOH and make
up the volume to one litre.
(ii) Manganous sulphate solution [MnSO4]- 480 gm MnSO4.4H2O / 400 gm
MnSO4.2H2O / 364 gmMnSO4.H2O is dissolved in distilled and dilute to 1000 ml.
(iii) Alkali-iodide azide reagent - 500 gm NaOH and 135 gm NaI are dissolved in
distilled water and dilute to 1000 ml. Add 10 gm NaN3 dissolved in 40 ml distilled
water. Or 700 gm KOH and 150 gm KI are dissolved in distilled water and dilute to
1000 ml. Add 10 gm NaN3 dissolved in 40 ml distilled water.
(iv) Starch solution (use an aqueous solution or soluble starch powder)- 2 gm
soluble starch and 0.2 gm salicylic acid as preservative in 100 ml hot distilled water.

Method:
• Take sample in BOD bottle add 1 ml MnSO4 solution followed by 1 ml alkali
iodide- azide reagent, yellow colour precipitate appears (if white precipitate
appears it indicates that there is no DO).
• Put stopper carefully to exclude air bubbles and mix by inverting bottle 40-50
times. Allow to settle the precipitate. When precipitation has settled sufficiently
add 1 ml conc. H2SO4. Mix by inverting bottle several times until precipitate
dissolved completely.
• Titrate 201 ml sample with standard 0.025N sodium thiosulphate solution to a
pale-yellow colour, add 1 ml starch indicator and continue titration until the
disappearance of blue colour.
• Note the reading and calculate DO by following formula

OBSERVATION TABLE:

Sr. Type of Sodium D.O.(mg/l)

thiosulphate
No. Sample used (ml)

Page 19 of 33
 Calculation:

D.O. (mg/L)= ml of Sodium thiosulphate used

➢ OVERALL RESULT:

➢ OVERALL CONCLUSION:

Page 20 of 33
Practical: 4 Determination of all the Biological Characteristics of the wastewater
sample

1. Biochemical Oxygen Demand (BOD)

Objective: To determine the BOD of given water sample.

Apparatus: BOD incubator, BOD bottle, Pipette, Burette, Conical flask, Volumetric
flask, Beaker, Measuring cylinder, Burette stand, and Pipette stand, Wash bottle.

Reagents:

(i) Phosphate buffer solution- 8.5 gm potassium dihydrogen phosphate (KH2PO4),

21.75 gm dipotassium hydrogen phosphate (K2HPO4) 33.4 gm. Disodium hydrogen
phosphate (Na2HPO4.7H2O) and 1.7 gm ammonium chloride NH4Cl are dissolved in
about 500 ml distilled water and dilute to 1000 ml. pH of the solution should be 7.2.
(ii) Magnesium sulphate solution [MgSO4.7H2O] - 22.5 gm MgSO4.7H2O is
dissolved in distilled water and dilute to 1000 ml.
(iii) Calcium chloride solution (CaCl2) - 27.5 gm CaCl2 is dissolved in distilled water
and dilute to 1 litre.
(iv) Ferric chloride solution [FeCl3.6H2O] - 0.25 g. FeCl3.6H2O is dissolved in
1000ml distilled water.
(v) Sulphuric acid [H2SO4] (1N)- Slowly and while stirring 28 ml conc. H2SO4 is
added in distilled water and dilute to 1000 ml.
(vi) Alkali sodium hydroxide [NaOH] (1N) - 40 gm NaOH is distilled water and dilute
to 1000 ml.
(vii) Glucose (Glutamic acid solution) - 150 gm glucose and 150 gm glutamic acid
are dissolved in distilled water and dilute to 1 litre. Prepare fresh immediately
before use.

Method:
• Preparation of dilution water.: Place desired volume of water in a suitable vessel
and 1 ml each of phosphate buffer, MgSO4, CaCl2, FeCl3, solution per litre of
water are added. Seed dilution water, if desired prepare 2-3 percentage of the
sample by dilution water and fill the 3 BOD bottles of each dilution. Also prepare
2% dilution of glucose-glutamic acid by dilution water and fill the 3 BOD bottles.
Fill 3 BOD bottles of dilution water as blank.
• Measure the dissolved oxygen of one of the three bottles of each dilution, of 2%
glucose-glutamic acid and blank, on starting day (0 day) by iodometric or
electrometric method.
• Keep the remaining bottles (2 bottles of each sample and blank) in the incubator
for 5 days if temperature is kept 20°C or for 3 days if temperature is maintained
at 27°C.
Page 21 of 33
 • After 5 or 3 days as the case may be measure the DO of the remaining bottles (2
bottles of each sample and blank).

OBSERVATION TABLE:

Sr. Type of Initial DO Final BOD.(mg/l)

DO
No. Sample

Calculation:

Calculate the BOD using following formula-

(a) When dilution water is not seeded:

BOD (mg/l) = (D1-D2)/p

(b) When dilution water is seeded:

BOD (mg/l) = [(D1-D2)-f x (B1-B2)]/p

Where,
D1= DO of diluted sample initially
D2 = DO of diluted sample after incubation.
P = Decimal volumetric fraction of sample used.
B1 = DO of seed control initially
B2 = DO of seed control after incubation
F = Ratio of % seed in diluted sample to % seed in seed control

RESULT:

CONCLUSION:

Page 22 of 33
 Practical: 5 Determination of all the Microbiological Characteristics of the
wastewater sample

1. Most Probable Number (MPN)

Objective: To determine the MPN of given water sample.

Apparatus- Laminar flow, Auto clave, Spirit lamp, Durham’s tubes, Test tubes, Cotton
bundle, Test tube stand, Pipette, Incubator.

Reagents- Chemicals for preparation of culture medium.

For preparation of 1 litre medium dissolve the quantity of all above chemicals by heating

do not boil.

Method:

Multiple tube fermentation (read the procedure carefully before analysis)

1. Prepare medium as per the procedure given above.

2. Fill 9 ml medium in washed test tubes carefully keep the tubes in test tube basket.

3. Fill the Durham tube with medium and put it in the test tube filled with medium.

4. Cap the test tube after putting Durham’s tube by cotton plug.
5. Cover test tube basket with aluminium foil (food grade) and also put required number of

10 ml pipettes for sterilization in the autoclave.

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6. Tighten autoclave cover put on autoclave when pressure is reached at 15 psi, keep on for
about 20 minutes at 15 psi keep the pressure at 15 psi by releasing steam (if required) by
steam release valve.

7. Put off autoclave after complete sterilization and open steam value for releasing
pressure.

8. After 30 minutes open autoclave and take out carefully the sterilized test tubes filled
with medium and other glass ware like pipettes.

9. Now test tubes when at normal temp keep in the test tube stand keeping 15 test tubes
for one sample in three groups of 5 test tubes each in the test tube stand with dilution
tubes.

10. Arrange test tubes so that for each sample 15 test tubes in 3 groups of 5 test tubes and
label the test tube stands as per sample codes.

11. Now take spirit lamps two numbers and put on the lamps at the table where test is
being performed if laminar air flow is available use that. Then there is no need for spirit
lamps.

12. Dilution of sample- There are different type of samples, which have different ranges of
coli forms dilution for some samples are given below-

13. After making proper dilution keep the sample tubes in bacteriological incubator for 24
hrs. at 35±0.5°C.
14. After 24 hrs check the tubes if there is air in the Durham’s tube also the colour of the

Page 24 of 33
media get changed light red if coliform is present due to fermentation of the media and
production of CO2 gas which gets filled in the Durham’s tube. These called positive tubes.
Count and note the number of positive tubes.

Reference Table-

Reference Table-

Page 25 of 33
Result:

The total coliform was found --------MPN/100ml in given water sample.

Page 26 of 33
Practical: 6 Testing a Soil Sample for all its parameters

1. Bulk Density

Objective - To determine the bulk density of given soil.

Apparatus- Oven, measuring cylinder and chemical balance.

Method- Dry the soil sample in oven at 105 0C until a constant weight is attained,
transfer a little dried soil to a measuring cylinder and note the volume. Record the
weight of this volume of soil on a balance.

OBSERVATION TABLE:

Sr. No. Weight of soil Volume Bulk density

of soil (gm/cm3)
(gm) (ml)

Calculation-

Bulk density (gm/cm3) = Weight of soil (gm)/Volume of soil (cm3)

Result- The bulk density of given soil was observed-------- gm/cm3.

2. Specific Gravity

Objective- To determine the specific gravity of given soil.

Apparatus- Oven, glass bottles, chemical balance.

Method-

Dry the soil in an oven at 1050C until a constant weight is attained. Fill a pre-weighted
glass bottle of known volume with dried soil and records its weight. Fill another pre-
Page 27 of 33
weighted glass bottle of same volume with distilled water and records its weight.

OBSERVATION TABLE:

Sr. Initial Weight of Initial Weight of Specific

weight of bottle with weight of bottle with gravity
No. soil (gm) bottle-2 distilled
bottle-
(gm) water (gm)
1(gm)

Calculation- Formula is given below:

Specific gravity = A2-A1/B2-B1

Where,
A2= weight of bottle with soil

A1= weight of empty bottle used for soil

B2= weight of bottle with distilled water

B1= weight of empty bottle used for distilled water

Result- The specific gravity of given soil was observed--------

3. Moisture Content

Objective- To determine the moisture content of given soil.

Apparatus- Oven and chemical balance.

Method-

Take a fresh homogenized sample of soil and weight it. Now dry it in an oven at 1050C until
a constant weight is attained. Cool in a desiccator and record the final weight of sample.

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OBSERVATION TABLE:

Sr. Initial weight of soil Weight of dried soil Moisture

(gm) (gm) content
No. (%)

Calculation- Moisture content (%) = I-F/I*100

Where, I= initial weight of soil

F= final weight of soil after drying

Result- The moisture content of given soil was observed ----------- %.

4. pH

Objective- To determine the hydrogen ion concentration (pH) of given soil.

Apparatus- Distilled water and pH meter.

Method-

Take 10 gm of air-dry soil/sediment and add 100 ml of distilled water to make a

suspension of 1:10 w/v dilution. Determine the pH of suspension.

OBSERVATION TABLE:

Sr. Sample description pH

No.

Calculation- (No calculation required. Note the reading directly from pH meter screen).

Result- The hydrogen ion concentration (pH) of given soil was observed ------ --.

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5. Electrical Conductivity

Objective- To determine the electrical conductivity of given soil sample.

Apparatus- Conductivity meter, distilled water, beaker.

Method-

Take 10 gm of air-dry soil/sediment and add 100 ml of distilled water to prepare

suspension (1:10 w/v). Then record the EC (Electrical Conductivity).

OBSERVATION TABLE:

Sr. Sample description Conductivity (µS)

No.

Calculation- (No calculation required. Note the reading directly from pH meter screen).

Result- The hydrogen ion concentration (pH) of given soil was observed ------ --.

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Practical: 7 Case Studies on Successful implementations of cleaner production

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Practical: 8 Case Studies on Waste Utilization from specific industries

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