Introduction and Technical Abstract

Purpose of Environmental Engineering Lab

Test the quality of air, water and soil as per the current environmental quality

standards. The testing procedure for water and waste water should be based on standard

methods 22nd edition published by American Public Health Association (APHA) and Water

Environmental Federation (WEF). Water standard can also be referred from Indian drinking

water standard BIS 10500-2012 and several other effluent standard can also be referred.

The important terms in laboratory uses are:

Amount of Substance onChemical ∈ g/lit

Molality =
Molecular weight

1M =1 Molar solution

Amount of Substance onChemical ∈ g/lit

Normality=
Equivalent weight

How to write the comments

1. Based on the result of the sample & evaluation, write whether it is accepted or

rejected.

2. According to its level of pollution, (i.e.) if it is in excess, write in detail about the

treatment method for reduction of excess amount of pollution.

3. Workout the cost of 1MLD of untreated water to convert it for drinking water.

4. Draw a neat flow diagram for the treatment process.

5. What are all the environmental and health effects of the pollutant when it is disposed

off untreatably and in case of consumption?

6. Explain about the new technologies to reduce the particular pollutants present in every

single analysis.

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 PHYSICAL PARAMETERS

COLOUR

GENERAL
Fiber in water may result from the presence of natural metallic ions (iron &
manganese), humus, oil, grease etc. The term Fiber is used in two ways i.e. True Fiber and
apparent Fiber. True Fiber is one in which turbidity has been removed and apparent Fiber
includes not only Fiber due to substances in solution but also due to suspended matter.

The Fiber of the sample may be observed usually through naked eyes after taking the
sample in a glass test tube.

RESULT

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 ODOUR

GENERAL
Odor is recognized by as a quality factor affecting acceptability of drinking water
tainting of fish and other aquatic organism aesthetics of recreational waters. Most organic and
inorganic chemicals contribute taste or odor. These chemicals may originate from natural
source such as decomposition of vegetable matter or from associated microbial activity and
from disinfectants of their products

The Odor may be qualitatively observed by smelling the water sample and record the
odor as normal, unpleasant. Fishy rotten egg etc;

RESULT

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 TEMPERATURE
GENERAL
Difference in water Temperature affects Dissolved oxygen, rate of photosynthesis
and metabolic rates of water life. The sensitivity of many organisms to toxic waste is also
influenced by water Temperature and Temperature change. It is important to note that
Temperature measurement is to be taken at the same time of the day, if readings are to be
compared.

PROCEDURE
1) Take the sample in a beaker.
2) Dip the Thermometer in the sample and note down the readings (º c)

RESULT

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 CONDUCTIVITY

AIM
To find out the conductivity of the given sample.

APPARATUS
Digital conductivity meter, sample, beaker, etc.

PRINCIPLE
The conductivity of the given solution is a measure of its ability to carry an electric
current and varies with number and type of ions present in it. In practice, measurement of
conductivity consists of measuring the resistance of column solution with reference to the
standard solution. Conductivity is expressed in form of Ms/cm.

OPERATING PROCEDURE
I. Before the instrument is switched ON, set control as follows.
(i) ON/OFF switch : Off position
(ii) RD/CAL switch : CAL position
(iii) RANGE switch : MID position
(iv)Temperature : Set the arrow at 25˚C
(v) CAL : MID position
(vi)Cell input : Connect wires from cell to these sockets, make
sure that main power card is not near or
crossing these wires.
II. GROUNDING
Ensure good grounding at the main socket.
1. Now connect the instrument to a 230V
single phase 3 pin 5 amp socket with
good grounding.
2. Switch ON the instrument (it should
display 100±200).
3. Allow to 10 minutes for warm up.

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 4. Adjust CAL till the display reads 1000
(neglect the decimal point).
5. If the instrument is being switched ON
for the first time or after a long idle
period or with a new conductivity, all the cell constant adjustment is to be
done, the procedure for this is given below.
a. Take the standard solution in a clean dry beaker an dip the cell into it
(it is recommended to use a constant temperature bath, so that the
temperature of the standard solution is maintained exactly at 20˚C).
b. Keep the RD/CAL switch in CAL position. Adjust the CAL
potentiometer to read 1000 on the display (neglect the decimal point).
c. Set the temperature (˚C) knob dial at 25˚C. (If the standard solution is
not being maintained at 25˚C, then set the dial or this control to the
temperature of the temperature of the standard solution). For best result
at 25±0.5˚C, atleast when first calibration is being carried out.
d. Set the RANGE switch to the proper range for the standard
solution.
e. Change the RD/CAL switch to RD position.
f. Adjust the cell constant ‘CELL K’ present control on the real panel till
the instrument reads the conductivity of the solution. Now the
instrument is ready and can be used to determine the specific
conductance/conductivity of the unknown solutions, for example with
a standard solution if 0.7453g of Kcal/kg solution. The control is
adjusted to give 1.409μscm-1 on display.
g. Temperature co-efficient ‘n’ is factorily adjusted for 2%/˚C as
this is the most common ‘n’ and applies to most of the solutions. For
solutions, where ‘n’ is not known, it has to be determined.

RESULT

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 TURBIDITY

AIM
To find out the turbidity of the sample.

APPARATUS
1) Nephelometric Turbidimeter
2) Nesslers tube

REAGENTS
1) Dissolve 1.0 gm of Hydrazine sulphate and dilute to 100 ml.
2) Dissolve 10 gm of hexamethylene tetramine and dilute to 100 ml.
3) Mix 5 ml of each of the above solution in a 100 ml volumetric flask and allow it to
stand for 25±3°c and dilute to 1000 ml. This solution has a turbidity of 40 NTU.

PROCEDURE
1) Switch on the Nephelometric turbidimeter and wait for some time till it warms up.
2) Set the instrument at 100 on the scale with 40 NTU standard suspension (In this case
every division on the scale will be equal to 0.4 NTU turbidity)
3) Shake the sample thoroughly, and keep it for sometime to eliminate the air bubble.
4) Dilute the sample with turbidity free water and again read the turbidity.
OBSERVATION AND CALCULATIONS

Sample Details Turbidity Remarks

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RESULT
DETERMINATION OF TOTAL DISSOLVED AND SUSPENDED
SOLIDS IN WATER
AIM
To determine total dissolved and suspended solids in the given water sample with the
stipulations as per IS: 3025 (Part 16 & Part 17).

INTRODUCTION
The term total dissolved solids refer to materials that are completely dissolved in water.
These solids are filterable in nature. It is defined as residue upon evaporation of filterable
sample. The term total suspended solids can be referred to materials which are not dissolved
in water and are non filterable in nature. It is defined as residue upon evaporation of non
filterable sample on a filter paper.

MATERIALS REQUIRED
1. Evaporating Dish
2. Water Bath
3. Oven
4. Desiccators
5. Analytical Balance
6. Graduated Cylinders
7. Dish Tongs
8. Gooch Crucibles
9. Filter
10. Vacuum Pumps
11. Crucible tongs
12. Forceps, Smooth –tipped

PROCEDURE
TESTING OF SAMPLE FOR TOTAL DISSOLVED SOLIDS
1. To measure total dissolved solids, take a clean porcelain dish which has been washed
and dried in a hot air oven at 180°C for one hour.

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 2. Now weigh the empty evaporating dish in analytical balance. Let’s denote the weight
measured as W1 = g.
3. Mix sample well and pour into a funnel with filter paper. Filter approximately 80 -100
mL of sample.
4. Using pipette transfer ‘x’ mL of unfiltered sample in the porcelain dish.
5. Switch on the oven and allowed to reach 105°C. Check and regulate oven and furnace
temperatures frequently to maintain the desired temperature range.
6. Place it in the hot air oven and care should be taken to prevent splattering of sample
during evaporation or boiling.
7. Dry the sample to get constant mass. Drying for long duration usually 1 to 2 hours is
done to eliminate necessity of checking for constant mass.
8. Cool the container in a desiccator. Desiccators are designed to provide an
environment of standard dryness. This is maintained by the desiccant found inside.
Don't leave the lid off for prolonged periods or the desiccant will soon be exhausted.
Keep desiccator cover greased with the appropriate type of lubricant in order to seal
the desiccator and prevent moisture from entering the desiccator as the test glassware
cools.
9. We should weigh the dish as soon as it has cooled to avoid absorption of moisture due
to its hygroscopic nature. Samples need to be measured accurately, weighed carefully,
and dried and cooled completely.
10. Note the weight with residue as W2 = g.

TESTING OF SAMPLE FOR TOTAL SUSPENDED SOLIDS

1. Place filtration apparatus with weighed filter in filter flask.
2. Mix sample well and pour into a graduated cylinder to the selected volume.
3. Apply suction to filter flask and seat filter with a small amount of distilled water.
4. Pour selected volume into filtration apparatus.
5. Draw sample through filter into filter flask.
6. Rinse graduated cylinder into filtration apparatus with three successive 10 mL
portions of distilled water, allowing complete drainage between each rinsing.
7. Continue suction for three minutes after filtration of final rinse is completed.
8. Dry filter in an oven at 103-105°C for at least 1 hour.
9. Cool filter in desiccator to room temperature.
10. When cool, weigh the filter and support.

CALCULATION
TABLE
Total Dissolved Solids
Description Weight (g)
Weight of the clean porcelain evaporating dish (g) W1
Weight of the dish and the residue (g) W2
Weight of residue(g) W

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 Volume of the Sample (mL) V
Total Dissolved Solids (mg/L) TDS

Weight of the clean porcelain evaporating dish (g) W1 =

Weight of the dish and the residue (g) W2 =
Weight of residue (g) W =
The volume of the sample (mL) V =

W 2−W 1 6
Total Dissolved Solids (mg/L) TDS = × 10
V

Total Suspended Solids

Description Weight (g)

Weight of the clean filter paper (g) W1
Weight of the filter paper and the residue (g) W2
Weight of residue(g) W
Volume of the Sample (mL) V
Total Suspended Solids (mg/L) TSS

Weight of the clean filter paper (g) W1 =

Weight of the clean filter paper and the residue (g) W2 =
Weight of residue (g) W =
Volume of the sample (mL) V =

W 2−W 1 6
Total Suspended Solids (mg/L) TSS = × 10
V

Results:

Total Dissolved Solids of the given sample =

Total Suspended Solids of the given sample =

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 TOTAL SOLIDS

AIM
To determine the total solids in the given water sample. Test procedure is in accordance to IS:
3025 (Part 15) - Reaffirmed 2003.

INTRODUCTION
The term “solids” is generally used when referring to any material suspended or dissolved in
water or wastewater that can be physically isolated either through filtration or through
evaporation.
Solids can be classified as either filterable or non filterable. Filterable solids may either be
settleable or non settleable. Solids can also be classified as organic or inorganic.
Total Solids is the term applied to the material residue left in the vessel after evaporation of a
sample and its subsequent drying in an oven at a defined temperature.
Measurement of Solids can be made in different water samples (industrial, domestic and
drinking water) and it is defined as residue upon evaporation of free water.
Thus, Total solids are nothing but summation of total dissolved solids and total suspended
solids.

APPARATUS
1. Crucible
2. Oven
3. Desiccators
4. Analytical Balance

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 5. Dish Tongs
6. Magnetic Stirrer
7. Wash Bottle

PROCEDURE
1. To measure total solids, take a clean porcelain dish which has been washed and dried
in a hot air oven at 105°C for one hour.
2. Now weigh the empty evaporating dish in analytical balance. Let’s denote the weight
measured as (W1).
3. Now we should have to decide what should be the volume of sample to be taken for
analysis.
4. Volume may be estimated either from values of specific conductance or general
thumb rule.
5. In general, select a sample volume that will yield residue between 2.5 and 200 mg
after drying.
6. Using pipette transfer 75mL of unfiltered sample in the porcelain dish.
7. Switch on the oven and allowed to reach 105°C. Check and regulate oven and furnace
temperatures frequently to maintain the desired temperature range.
8. Place it in the hot air oven and care should be taken to prevent splattering of sample
during evaporation or boiling.
9. Dry the sample to get constant mass. Drying for long duration usually 1 to 2 hours is
done to eliminate necessity of checking for constant mass.
10. Cool the container in a desiccator. Desiccators are designed to provide an
environment of standard dryness. This is maintained by the desiccant found inside.
Don't leave the lid off for prolonged periods or the desiccant will soon be exhausted.
11. Keep desiccator cover greased with the appropriate type of lubricant in order to seal
the desiccator and prevent moisture from entering the desiccator as the test glassware
cools.
12. We should weigh the dish as soon as it has cooled to avoid absorption of moisture due
to its hygroscopic nature.
13. Samples need to be measured accurately, weighed carefully, and dried and cooled
completely.
14. Note the weight with residue as (W2).

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OBSERVATION

Description Weight (g)

Initial Weight of the Crucible (g) W1
Final Weight of the Crucible + sample (g) W2
Weight of residue(g) W
Volume of the Sample (mL) V
Total Solids (mg/L) TS

CALCULATION

W 2−W 1 6
Total solids (mg/l) = × 10
V

RESULT
The Total Solids of the given sample is

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DETERMINATION OF TOTAL VOLATILE SOLIDS (ORGANIC) AND
TOTAL FIXED SOLIDS (INORGANIC) IN WATER

AIM
To determine total organic and inorganic solids in the given water sample with the
stipulations as per IS: 3025 (Part 18) - Reaffirmed 2002.

INTRODUCTON
The term total volatile solids refer to materials that are completely volatilised from water at
higher temperature (550ºC). These solids are often referred to the organic content of the
water. The term total fixed solids can be referred to materials which are not volatilised from
water at higher temperature (550ºC). These solids are often referred to the inorganic content
of the water.
APPARATUS REQUIRED
1. Evaporating Dish
2. Water Bath (Steam Bath)
3. Oven
4. Desiccators
5. Weighing balance
6. Dish Tongs
7. Magnetic Stirrer
8. Wash Bottle

PROCEDURE

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TESTING OF SAMPLE
1. To measure total volatile solids and fixed solids, take a clean silica crucible which has
been washed and dried in a hot air oven at 105ºC for one hour and ignited at 550ºC to
remove all organic materials present in it.
2. Now weigh the empty silica crucible in analytical balance. Let’s denote the weight
measured as W1 = g
3. Using pipette transfer 75mL of unfiltered sample in the porcelain dish.
4. Switch on the oven and allowed to reach 105°C. Check and regulate oven and furnace
temperatures frequently to maintain the desired temperature range.
5. Place the silica crucible in the hot air oven and care should be taken to prevent
splattering of sample during evaporation or boiling.
6. Dry the sample to get constant mass. Drying for long duration is done to eliminate
necessity of checking for constant mass.
7. Cool the container in a desiccator. Desiccators are designed to provide an
environment of standard dryness. This is maintained by the desiccant found inside.
8. Don't leave the lid off for prolonged periods or the desiccant will soon be exhausted.
9. We should weigh the dish as soon as it has cooled to avoid absorption of moisture due
to its hygroscopic nature.
10. Samples need to be measured accurately, weighed carefully, and dried and cooled
completely.
11. Note the weight with residue as W2 = g
12. Switch on the furnace and allow it to reach 550°C. Check and regulate the furnace
temperatures frequently to maintain the desired temperature range.
13. Place the silica crucible in the furnace and care should be taken while keep the
crucible inside the furnace since it will be too hot.
14. Allow it to ignite for 20 minutes to get constant mass.
15. As above, cool the silica crucible in a desiccator to room temperature.
16. Weigh the dish as soon as it has cooled to avoid absorption of moisture due to its
hygroscopic nature.
17. Note the weight with residue as W3 = g

TABLE
Total Volatile Solids
Description Weight (g)

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 Weight of the clean silica crucible (g) W1
Weight of the silica crucible and the residue (g) W2
Weight of residue (g) W
Weight of the silica crucible and the ash (g) W3
Weight of ash (g) W
Volume of the Sample (mL) V
Total Volatile Solids (mg/L) TVS

Calculation
Total Volatile Solids
Initial weight of the evaporating dish + sample (W1) = ……….. g
Final weight of the evaporating dish + sample after drying at 105ºC (W2) = ……….. g
Final weight of the evaporating dish + sample after drying at 550ºC (W3) = ……….. g
Weight of volatile substance (W) = W2 – W3 g

W 2−W 3 6
Total Volatile Solids = × 10
V
= mg/L

TABLE
Total Fixed Solids
Description Weight (g)
Weight of the clean silica crucible (g) W1
Weight of the silica crucible and the residue (g) W2
Weight of residue (g) W
Weight of the silica crucible and the ash (g) W3
Weight of ash (g) W
Volume of the Sample (mL) V
Total Fixed Solids (mg/L) TFS

Calculation
Initial weight of the evaporating dish (W1) = ……….. g

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Final weight of the evaporating dish + sample after drying at 105ºC (W2) = ……….. g
Final weight of the evaporating dish + sample after drying at 550ºC (W3) = ……….. g
Weight of non volatile substance (W) = W3 – W1 g

W 3−W 1 6
Total Fixed Solids = × 10
V
= mg/L

RESULTS
In the given sample, Total volatile solids is equivalent to mg/L.
Total fixed solids is equivalent to mg/L.
DETERMINATION OF SETTLEABLE SOLIDS
AIM
To determine the amount of settleable solids present in the given sample.

PRINCIPLE
Suspended solids in waste water include both settleable and non settleable solids.
Solids which settle down in a reasonable time (one hour) are called settleable solids. To
determine this, a device known as Imhoff cone is used. It is a conical glass jar of 1 litre
capacity with graduations on the wall. The settled solids will be collected at the apex of the
cone whose volume can be noted on the graduation.

APPARATUS
Imhoff cone, measuring jar, stand, glass beaker.

PROCEDURE
Fill the Imhoff cone up to the 1 litre mark with a mixed sample and allow settling
after about 20 minutes. Gently dislodge any deposit on the side of the vessel by spinning the
cone so that all solids will be collected at the apex of the cone. After one hour note the
volume of the settled matter and express the results as ml settleable solids per litre.

RESULT
Concentration of settleable solids in the given water sample = ____________ ml//L

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OBSERVATION
Lower Value = __________
Upper Value = __________
Amount of settleable solids in the Imhoff cone =

JAR TEST FOR COAGULATION

AIM
To determine the optimum dosage of coagulant required for the given water sample.
PRINCIPLE
Optimum dosage of coagulant is the minimum amount of coagulant required to
produce a good flog. Filter alum (Aluminum sulphate) is represented by the formula
A12(SO4)3.14H2O.Aluminum sulphate after dissolving in water will dissociate into positively
and negatively charged particles. The positively and negatively charged particles. The
positively charged particles neutralize the negatively charged colloidal particles which is
available in the form of turbidity neutralization results in formation of floc and agglomeration
of particles. Finally particle will increase in their size and settle down at the bottom due to
gravity.
APPARATUS
Jar Test apparatus, beaker and 10ml micro pipette.
REAGENTS
Dissolve 3g of alum in distilled water and make up to 100ml
PROCEDURE
Jar Test apparatus consist of a rotary device with 6 Nos, of stirring rods provided with
pedals at lower ends. Speed of the stirrer can be adjusted by means of a driving mechanism.
Take 500ml of sample in each jar. Add different amount of standard alum solution in
the range of 0.5ml, 1ml, 1.5ml, 2ml and 3ml. The stirrers are to be immersed in the jar such
that it should not touch the sides or bottom of the jar. The driving unit is started. The stirrers

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are allowed to run at rapid speed for first 2 to 3 minutes, and then at slow speed for 15
minutes. The driving unit is then stopped. The jars are kept and note the settling for 30
minutes. Observe the jars and note the settling of sediments. Note down the minimum dosage
of coagulant that produces that produces good and visible floc. Tabulate the values. Repeat
the experiment if necessary to narrow down the range and find out the minimum amount of
alum required for satisfactory coagulation.
Repeat the same iron alum coagulant =________ mg/L
RESULT
i. The optimum dosage of alum as coagulant =________ mg/L
ii. The optimum dosage of Ferric sulphate as coagulant =________mg/L

CHEMICAL EXAMINATION OF WATER SAMPLE

MEASUREMENT OF pH
AIM
To determine the pH of given sample.
PRINCIPLE
Measurement of pH is one of the most important and frequently used testes in water
chemistry. pH of a solution is defined as the negative logarithm of hydrogen ion activity at a
given temperature. The intensity of acidic or basic character of the solution is indicated by pH
or hydrogen ion activity. pH scale represents a range of hydrogen activity from 0 to 14, with
7 pH representing the absolute neutrality. Values of pH lower than 7 indicate the hydrogen
ion concentration is greater than hydroxide the hydrogen ion concentration and the water is
termed acidic. The opposite condition is implied when pH exceeds 7, and the water is termed
basic. The basic principle of electronic measurement is determination of the activity of
hydrogen ions by potentiometer measurement using a standard hydrogen electrode (glass
electrode) and a reference electrode. When these electrodes are dipped in an aqueous solution
they generate and EMF (electromotive force) proportional to the pH of the solution. The
magnitude of the EMF is also depended on the temperature of the solution.
APPARATUS
pH meter, Reference electrode, glass electrode, Beaker and stirrer.
OPERATING PROCEDURE
1. Proper conditioning of the glass electrode must be done.

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 2. Keep the main Switch OFF
3. Keep the mode switch in STD BY
4. Connect the electrode jacks in their respective sockets.
5. Now insert the power plug to a 3 pin 5 amps socket of 230V, 50 Hz single phase, with
good grounding. If grounding is not proper, connect separate load from the GND
terminal, which is on the back panel to a good ground.
6. Switch on the instrument. It should read 000. If it is not reading zeros, keep it on for
few minutes.
7. Let the instrument stabilize for about 10 minutes. In case the instrument displays
some reading other than 000 in the STD BY position of the mode switch on lamp of
more than 100W over the instrument to evaporate the moisture deposited on the
instrument for about 10-15 minutes or till the instrument display 000 in the STD BY
position.
8. Take a fresh buffer solution of 7 Ph. Dip the electrode in the solution after thoroughly
washing the electrode with distilled water and dry with filter paper or tissue paper. Set
the mode switch to mode mV position. It should be zero mV, if it is a correct 7 pH
buffer solution. In case if it does not read mV, adjust the standardise knob to get 0 mV
mode. Once it reads the zero mV it will read pH7 by setting the mode switch to zero
pH position.
9. Rinse the electrode in distilled water. Wipe with the tissue paper and dip in fresh 4 pH
buffer solution. Set the temperature knob to the solution temperature. Set the mode
switch to pH. The instrument should display 4 pH if pH does not adjust the “slope
adjustment” control it with an insulated tip screwdriver till the instruments reads pH
4.
10. Keep the CHK/RD switch CHK position along with mode switch pH position and set
the temperature. The instrument should be 4 pH. Then adjust with insulated
screwdriver. The control CHK-CAL which is on the back panel till the instrument
reads pH 4.

Measurement of pH
1. Wash the electrode with distilled water and wipe it filter pH tissue paper.
2. Dip the electrode in the sample solution.
3. Keep the temperature knob at 25℃

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 4. Set the CHK/RD switch in CHK position and mode switch in pH position. The
reading should be 4 pH.
5. Set the CHK/RD switch from CHK to RD position.
6. Adjust the temperature knob to the sample temperature. The reading will be the pH of
thee samples solution within+0.01 Ph.
7. For the measurement of mV set the mode switch to mV position. The reading will be
the polarity indication.
8. Slope the adjustment and the CHK-CAL adjustment should be done only when it is
must. They are factory calibrated.

RESULT
The pH of the given sample, Sample (1) = __________
Sample (2) = __________
DETERMINATION OF ALKALINITY

AIM
To determine the alkalinity present in the give water sample, and in Tap water.
PRINCIPLE
The alkalinity of a water sample is a measure of its capacity to neutralize acids.
Alkalinity is due to the basic constituents of water which can be titrated with a strong mineral
acid. It is caused by three major species namely, hydroxides, carbonates, and bicarbonates. It
is common practice to express alkalinity as phenolphthalein alkalinity and total alkalinity.
Phenolphthalein alkalinity is a measure of hydroxides and carbonates in water sample and
total alkalinity refer to the amount of acid required to react with hydroxide, carbonate and
bicarbonate present in water sample. Alkalinity is usually expressed in terms of CaCO3
(mg/l)
REACTIONS
Na2 CO3 + HCL → NaCl + NaHCO3
NaHCO3 + HCL → NaCl + CO2 + H2O
Na2CO3 + 2HCl → 2NaCl + H2O + CO2
APPARATUS
Burette, pipette, conical flash, standard flash and funnel.
REAGENTS
1. Standard Hydrochloric acid

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 8.3 ml HCL in 100 ml of distilled water.
2. Standard sodium Carbonate solution (0.05)
Dissolve 5.3 g of sodium carbonate in 1000 ml of distilled water.
3. Phenolphthalein indicator
Dissolve 5 g solid in 500ml alcohol (ethyl or isopropyl) and add 500 ml distilled
water.
4. Methyl orange indicator
Dissolve 0.5g Solid in 1 litre of distilled water.

PROCEDURE
1. Standardization of HCL
Frist, burette is washed with water and then rinsed with HCL. Then HCL is filled in
the burette up to zero mark. 20ml of standard Na2O3 is pipetted out into a clean conical flask
and one drop of methyl orange indicator is added. The solution is titrated against HCL taken
in the burette. End point is the appearance of wine red colour. Titration is repeated for
concordant values.

2. Estimation of Alkalinity
i) Phenolphthalein Alkalinity (Carbonate Alkalinity)
Pipette out 20 ml of sample in the conical flash and add one drop of phenolphthalein
indictor. Titrate with HCL taken in the burette. The end point is the disappearance of pink
colour.
ii) Total Alkalinity
Add one drop methyl orange indicator to the solution in which the phenolphthalein
alkalinity has been determined. Titrate against the standard acid. The end point is given by
the change of colour from yellow to pink. Report the titration for concordant values.

OBSERVATION TABLES
1. Standardization of HCL
HCL VS Na2Co3
Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

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CALCULATION
Volume of Na2 CO3 Solution V1 = 20ml
Number of mole of Ma2 CO3 N1 = 1
Molarity of Na2 CO3 M1 = 0.05M
Volume of HCL V2 = ml
Number of mole of Hcl N2 = 2
Molarity of HCL M2 = ?
(V 1 M 1) ( V 2 M 2)
=
N1 N2

M2 = {(V1 M1 / N1) × (N2 / V2)}

= M
2. Estimation of Water Sample
HCL vs Water sample

Burette reading Concordant reading

Burette Pipette Final(ml)
Sl. Methyl
Solution Solution Initial Methyl Phenolthalein
No Phenolthalein Orange
(ml) (ml) (ml) Orange (ml)
end point (ml)
end point

Table A. Concentration of Carbonates, Bicarbonates and Hydroxyl ions

Sl. No Result of Titration (OH-) ion Carbonate ion Bicarbonate

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 (CO3-) ion (HCO3-)

P=0
1 0 0 M

P=M
2 M 0 0

1
3 P= 2 M 0 2P 0

1
4 P< 2 M 0 2P M-2P

5 1 2P-M 2(M-P) 0
P> 2 M

OBSERVATION AND CALCULATIONS

For given water sample
P= ml ; M= ml
Compare P and M in Table A and get VHCl for OH-, CO- and HCO3- end points (whichever is
applicable) in terms of P and M.
i. Hydroxyl Alkalinity (OH-)

VHCl = V2 = ------- ml (as per Table A)

V1 = 20 ml
N1 = N2 = 1
(V1 M1)/N1 = (V2 M2) / N2
M1 = {(V1 M2 / N1) × (N2 / V2)} = --------- M
Hydroxide Alkalinity = M1 × (Molecular weight of OH-) × 1000
= M1 × (17) × 1000
= ---------- mg/L
ii. Carbonate Alkalinity (CO3-)

VHCl = V2 = ------- ml (as per Table A)

V1 = 20 ml
N1 = N2 = 1

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 (V1 M1)/N1 = (V2 M2) / N2
M1 = {(V1 M2 / N1) × (N2 / V2)} = --------- M
Carbonate Alkalinity (CO3-) = M1 × (Molecular weight of CO3-) × 1000
= M1 × (60) × 1000
= --------- mg/L

iii. Bicarbonate Alkalinity (HCO3-)

VHCl = V2 = ------- ml (as per Table A)

V1 = 20 ml
N1 = N2 = 1
(V1 M1)/N1 = (V2 M2) / N2
M1 = {(V1 M2 / N1) × (N2 / V2)} = --------- M
Bicarbonate Alkalinity = M1 × (Molecular weight of HCO3-) × 1000
= M1 × (61) × 1000 = --------- mg/L

RESULTS
1) Phenolphthalein Alkalinity of given water sample = __________________ mg/L
2) Total Alkalinity of given water sample = __________________ mg/L
3) Phenolphthalein Alkalinity of tap water = __________________ mg/L
4) Total Alkalinity of tap of tap water = __________________ mg/L

Page 25 of 66
 DETERMINATION OF TOTAL HARDNESS
(EDTA METHOD)
AIM
To estimate the amount of total hardness present in the given water sample.

PRINCIPLE
Hardness is caused by divalent metallic cations. Such ions are capable of reacting
with soap to form precipitates and certain anions present in the water to form scale. The
principle hardness causing cations are calcium, magnesium, ferrous iron and manganese ions.
EDTA method involves the use of solutions of ethylene diamine tetra acetic acid (EDTA) by
its sodium salt as the titrating agent. The compounds usually represented by EDTA form
stable complex ions with hardness causing ionCa2+¿ ¿, Mg 2−¿¿ represented by the equation.
2−¿¿
M + EDTA -----------> (M EDTA) COMPLEX

The dye “Eriochrome Black T” serves as an excellent indicator to show when all the
ions causing hardness have been completed. When a small amount of “Erichromme Black T”
is a added to hard water it forms wine red complex with Ca2+¿ ¿, and Mg 2−¿¿ ions represented
by the equation.

Page 26 of 66
 2−¿¿
M + Eriochrome Black T -----------> (Mg Eriochrome Black T) wine red complex

During the titration with EDTA, all the free hardness ions are complexes according to
equation (1). Finally EDTA disrupts the red (M-Eriochrome Black T) complex because it is
capable of forming a more stable complex with hardness ions. This action frees the
Eriochrome Black T indicator and the wine red colour change to a distinct blue colour,
indicating the end of the titration.

APPARATUS
1. Burette, pipette,
2. Conical flask,
3. Standard flask and
4. Funnel.

REAGENTS
1) EDTA Solutions
Dissolve 3.4g of the di-sodium salt of EDTA in distilled water and dilute to 1 litre.
Store in polythene container.
2) Buffer Solution
Dissolve 67.5g of ammonium chloride NH 4CL and 570ml of NH 4OH (ammonium
hydroxide) and dilute to 1litre. Keep in tightly stoppered bottle.
3) Eriochrome Black T
Dissolve 0.5g of solid in 100ml of ethanol or isopropyl alcohol.
4) Standard Hardwater
Dissolve 1g of pure dry calcium carbonate in concentrated hydrochloric acid and
make it up to 1 litre.

PROCEDURE
1) Standardization of EDTA
Pipette out 20ml standard hard water into a clean conical flask and add 1ml of buffer
solution and mix. Add 1 drop of indicator solution and titrate with standard EDTA(0.01M)

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till wine red colour changes to blue. Note down the volume of EDTA required. Repeat the
titration for concordant values.
2) Estimation of total hardness
Pipette out 20ml of given sample into a clean conical flask and add 1 of buffer
solution and mix. Add 1 drop of indicator solution and titrate with standard EDTA till wine
red colour changes to blue. Note down the volume of EDTA required. Repeat the same
procedure with tap water to get the concordant values.

INFERENCE
If metal ions are present they interfere with the end point of titration. Inhibitors are
added to eliminate this phenomenon. Dissolve 4.5g of hydro oxylamine hydrochloride in
100ml ethyl alcohol (95%), 1ml of the sample followed by 1 ml of buffer before titrating with
EDTA.

OBSERVATION TABLES
1. Standardization of EDTA
EDTA Vs Hard water
Indicator: Eriochrome Black-T
Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of hard water =

Volume of EDTA for hard water =

Page 28 of 66
2. Estimation of total hardness
i) For tap water
EDTA Vs Tap water
Indicator Eriochrome black-T
Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of EDTA for tap water =

ii) For given sample

EDTA Vs Given Sample
Indicator: Eriochrome black-T
Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of EDTA for given sample =

CALCULATIONS
(i) For Given Sample

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 (Volume of EDTA for given sample )
Total hardness = x1000 mg/l as CaCo 3
(volume of EDTA for hard water )
(ii) For Tap Water
(Volume of EDTA for tap water )
Total hardness = x1000 mg/l as CaCo 3
( volume of EDTA for hard water )

RESULTS
Total hardness of the given water sample =________________mg/L
Total hardness of the tap water = ________________mg/L

DETERMINATION OF CHLORIDE
(ARGENTOMETRIC METHOD)
AIM
To determine the amount of chlorides present in the given sample.
PRINCIPLE
Chloride, in the form of chloride (cl-) ion is one of the major in organic anions in
water and wastewater, chloride above 250mg/l concentration give a salty rests to water,
which is objectionable from the public. They can be measured by means of volumetric
procedures using internal indicators. For most purpose the Mohr method (Argent metric
method) using silver nitrate as titrate and potassium chromate as the indicator is found
satisfactory.
AgNO3 + NaCl AgCl + NaNO3
K2CrO4 + 2 AgNO3 Ag2CrO4 + KNO3
As per the above reaction, Silver Nitrate reacts with chlorides present in the given
sample and forms white colour silver chloride (AgCl) precipitate. When all the chlorides are

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neutralized the addition of excess silver nitrate precipitate as silver chromate (Ag 2CrO4). This
indicates the end point of titration.
APPARATUS
Burette, pipette, conical flask, wash bottle, weight box
REAGENTS
i) Standard silver nitrate solution (0.01M)
Dissolve 1.698 g AgNO3 in distilled water and dilute to 1000 ml.
ii) Potassium chromate solution (Indicator)
Dissolve 5g Potassium chromate in 100ml of distilled water.

PROCEDURE
1. Titration of silver nitrate with blank solution
Pipette out 20ml of distilled water into a clean conical flask. Add 2 to 3drops of
potassium chromate and the colour of the solution turns to yellow. Then the solution is
titrated against silver nitrate taken in the burette. The end point is the appearance of reddish
lint. Repeat the titration for concordant value.
2. Estimation of chlorides in the given sample
Pipette out 20ml of given sample into a clean conical flash and add 2 to 3 a drops of
potassium chromate. Titrate the yellow coloured solution against the silver nitrate in the
burette. End point is the appearance of reddish lint. Repeat the procedure to get concordant
values. Similarly repeat the same procedure with tap water to get concordant values.

Titration 1: AgNO3 Vs Distilled Water

Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of AgNo3 for distilled water (B) =________________

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Titration 2: AgNO3 Vs Given sample 1
Burette Pipette Burette Reading Concordant
Sl.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of AgNO3 for sample (A) = ________________

Volume of AgNO3 for distilled water (B) =________________

Titration 3: AgNO3 Vs Given sample 2

Burette Pipette Burette Reading Concordant
S.No.
Solution(ml) Solution(ml) Initial(ml) Final(ml) Value (ml)

Volume of Pipette solution =________________

Volume of AgNO3 =________________

CALCULATION

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Volume of AgNO3 for sample (A) = ________________
Volume of AgNO3 for Distilled Water (B) = ________________

Amount of chloride present = (A-B) x Molarity of AgNO3 x At. Wt. of Cl x 1000

Sample Volume

=________________ mg/L

RESULT
i) Amount of chloride present in given sample = ________________mg/L
ii) Amount of chloride present in tap water = ________________mg/L

DETERMINATION OF RESIDUAL CHLORINE

(STARCH –IODIDE METHOD)
AIM
To determine the amount of residual chlorine present in the given sample.
PRINCIPLE
Drinking water is chlorinated either with free chlorine or with hypochlorite’s for
disinfections. Therefore it contains residual chlorine, i.e excess chlorine not consumed by
pollutants in water. Starch-oxide method for determination of chlorine depends upon the
oxidizing power of free and combined residual chlorine in water to convert iodide ion to free
chlorine. This oxidation is represented as
Cl2+2I- I2+2Cl-
I2 + 2Na2SO4 Na2S2O6 + 2NaI
I2+ Starch Blue Colour
The iodine released is chlorine with a standard solution of Sodium Thiosulphate. The
end point is the disappearance of blue colour.
APPARATUS REQUIRED

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 Burette, pipette, conical flask, standard reagent wash bottle, Weight box
REAGENTS
1. Standard Sodium Thiosulphate
Dissolve 5.5g of Sodium thiosulphate in one litre of distilled water
2. Potassium Dichromate
Dissolve 0.3g of Potassium Dichromate in 500 ml of distilled water
3. Potassium Iodide
Dissolve 20g of Potassium Iodide in 200 ml of distilled water
4. Hydrochloric acid
Dissolve 150 ml of conc. Hydrochloric acid in one litre of distilled water
5. Starch Indicator
Take 0.5g of starch prepare with water & make 100 ml of water and boiled by stirring
and finally cooled to room temperature.

PROCEDURE
1. Standardization of Sodium Thiosulphate
Pipette out 20 ml of Potassium Dichromate in a clean conical flask. Add 10-20 ml of
Hydrochloric Acid and 5ml of Potassium Iodide. After one minute add 3 drops of Starch and
the colour of the solution turns to dark blue. Titrate the solution against Sodium Thiosulphate
taken in the burette. End point is the change of colour from wine red to blue. Repeat the
titration for concordant values.

2. Estimation of Residual Chlorine

Take given sample of water in 200 ml bottle and add 5ml of KI. Add drops of Starch
shake the bottle and keep it for 5 minutes without disturbance. Take these contents in a
conical flask and titrate it against Sodium thiosulphate taken in the burette. End point is the
change of colour from blue to colourless. Repeat the titration for concordant values.

1. Standardization of Na2S2O3
Indicator: Starch

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 Burette Reading
Burette Pipette Concordant
Sl.No
Solution(ml) Solution(ml) Value (ml)
Initial(ml) Final(ml)

Volume of K2Cr2O7 (V1) =

Normality of K2Cr2O7 (N1) =
Molarity of K2Cr2O7 (M1) =
Volume of Na2S2O3 (V2) =
Normality of Na2S2O3 (N2) =
Molarity of Na2S2O3 (M2) =
V 1 MI N 2
Molarity of Na2S2O3 (M2) = ×
N1 V2
= __________ M

2. Estimation of Residual Chlorine

For Sample
Indicator: Starch

Burette Reading
Sl.N Burette Pipette Concordant
o Solution(ml) Solution(ml) Value(ml)
Initial(ml) Final(ml)

Volume of Sample water =

Volume of Na2S2O3 =

CALCULATIONS

Page 35 of 66
 Amount of Residual Chlorine = 35.45× Volume of Na2S2O3 × M2 ×1000 mg/L
Volume of Sample

RESULT
Amount of residual chlorine present in the given sample of water = ......... mg/L

DETERMINATION OF DISSOLVED OXYGEN

(WINKLER’S METHOD)
AIM
To determine the amount of dissolved oxygen present in the given water sample.
PRINCIPLE
Dissolved oxygen levels in natural and wastewater depends on the physical, chemical
and biological activities in the water body. The analysis of dissolved oxygen is a key test in
water pollution and waste treatment process control. In Winkler’s method of DO
determination, dissolved oxygen present in water oxidize Mn 2+ to Mn2+ under alkaline
conditions and that manganese in the higher state of valence oxidizer iodide to iodine 2I- to
I2 under acid conditions. The iodine thus liberated is titrated with sodium thiosulphate
solution using starch as the indicator.
EQUATIONS
Mn2+ + 2OH- + (O) MnO2 + H2O
(DO)

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 MnO2 + 2I- + 4H+ Mn2+ + I2 + 2H2O
I2 + 2Na2S2O3 Na2S4O6 + 2NaI
APPARATUS
Burette, Pipette, conical flash, weight box, balance, reagent bottle, distilled water
bottle.
REAGENTS
1. Sodium thiosulphate Solution
Dissolve 6.2g of sodium thiosulphate crystals and dilute to one litre. Preserve by
adding 1ml chloroform and 1g NaOH per litre of solution.
2. Potassium Dichromate
Dissolve 0.3g of potassium dichromate in 500 ml of distilled water.
3. Potassium Iodide
Dissolve 10g of potassium iodide in 100 ml of distilled water.
4. Hydrochloric acid
Dissove 150 ml of HCl in 1 litre of water.
5. Manganese Sulphate solution
Dissove 50g MnSO4 in 100 ml of distilled water

6. Alkaline Potassium Iodide

Dissolve 70 g of potassium hydroxide in 40 g of NaOH and 15g of potassium iodide
in 100 ml of distilled water.
7. Starch Indicator
Take 0.5 g of starch prepare a paste with water & make 100 ml of distilled water and
boil by stirring and finally cool to room temperature.

PROCEDURE
1. Standardization of sodium thiosulphate solution
Take 20ml of potassium dichromate and pipette out in a clean conical flask. Add 10 to
12ml of HCl and 5ml of KI and add 3 drops of starch. Titrate this against sodium thiosulphate
taken in the burette. Now the solution in the conical flask becomes greenish in colour. End
point is the appearance of colorless solution. Repeat the titration of concordant values.

2. Estimation of dissolved oxygen

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 Fill up 300 ml reagent bottle with water sample leaving about 10ml space at the top.
Add 4 to 5 ml of MnSO4 and 3-4 ml of alkaline potassium iodide. This results in the
formation of manganese hydroxide precipitate. Fill the gap with water and close the bottle.
Keep it without any disturbance for 10 minutes. Add conc. H 2SO4 to dissolve manganese
oxide. Now the colour of the solution turns to yellow. Take 100 ml of solution in a clean
conical flask and add 2-3 drops of starch and titrate this yellow colored solution against
sodium thiosulphate taken in the burette. End point is the appearance of colorless solution.
Repeat the titration for concordant values.

OBSERVATION
Standardization of Sodium Thiosulphate Solution

Burette Pipette Burette Reading Concordant

Sl.No.
Solution(ml) Solution(ml) Value(ml)
Initial(ml) Final(ml)

Volume of K2Cr2O7 (V1) =

Molarity of K2Cr2O7 (M1) =
No. of moles of K2Cr2O7 (N1) =
Volume of Na2s2O3 (V2) =
Molarity of Na2S2O3 (M2) =
No. of moles of Na2S2O3 (N2) =
Molarity of Na2S2O3 (M2) = V1M2 x N2
N1 x V2
Molarity of Na2S2O3 (M2) = __________ M

Estimation of Dissolved Oxygen for sample

Indicator: Starch
Burette Pipette Burette Reading
Sl.No. Concordant Value(ml)
Solution(ml) Solution(ml) Initial(ml) Final(ml)

Page 38 of 66
Volume of water sample = ________________ml.
Volume of Na2S2O3 = _________________ ml.

CALCULATION
D.O present in the tap water = 8 x Volume of Na2S2O3 x M2 x 1000
_______________________________
Volume of Sample

RESULT
Concentration of dissolved oxygen present in the given sample = _______________
mg/L.

DETERMINATION OF MANGANESE
(PERIODATE METHOD)
AIM
To determine the amount of manganese iron in the given water sample.
PRINCIPLE
Manganese occurs in domestic waste water, industrial effluent and receiving streams.
Manganese present in the ground water is usually in soluble divalent ionic form due to lack of
oxygen. Such water exposed to atmosphere turn turbid because of the oxidation and become
unacceptable to domestic use. In addition to toxic nature, manganese imparts objectionable
taste and colour to laundry and plumbing fixtures on the even in low temperature (0.05mg/l).
Manganese allowed in the drinking water is 0.3mg/l. It also interferes on the distribution
system. Per iodate method involves the oxidation of manganese compounds to permanganate
ion by potassium per iodate in the presence of strong acids. The colour developed is stable for

Page 39 of 66
at least 24 hours is the presence of excess potassium per iodates and the corresponding
concentration is found out using graph after complete analysis.
APPARATUS
Colorimeter, nesslers tubes, acid washed glassware.
REAGENTS
1. Concentrated sulphuric acid.
2. Concentrated phosphoric acid (85%) & 15% of distilled water.
3. Concentrated nitric acid.
4. Preparation of blank/stock solution:
Dissolve 0.287 g of KMNO4 in 100ml of distilled water. This is the stock
solution and is equivalent to 1000 ppm.
PROCEDURE
1. Preparation of calibration curve
a) Take 10 ml of 1000 ppm solution and dissolve it in distilled water and make upto 100
ml. This will be equal to 100 ppm.
b) Take 10 ml of the 100 ppm solution and dissolve it in distilled water and make
upto100 ml. This will be equal to 10 ppm solution.
c) Similarly prepare 20 ppm to 10 ppm standards.
d) The colour intensity of each of the standard is measured in terms of absorbance value
at wavelength of 525 nm in the colorimeter.
e) A linear line is obtained by plotting concentration of standards on the x axis and
absorbance on the Y axis.

2. Preparation of water sample and blank

Take 100 ml of sample in a conical flask. Add 5 ml of H 2SO4, 5 ml of HNO3 and 5 ml
H3PO4 to the sample. The solution is heated to boiling point and cooled to room temperature.
Then 0.3g potassium per iodate is added and a colour complex is developed. The absorbance
value of the colour complex is measured in the colorimeter. A blank is prepared in the same
manner as the standards.

3. Determination of unknown concentration of water sample

Page 40 of 66
 The absorbance value of the blank is set in the colorimeter. Then the absorbance value
of the water sample is interpolated in the calibration curve and the corresponding
concentration of manganese in the water sample is measured.

OBSERVATION
Table 1: Data for calibration curve
Sl.NO CONCENTRATION ABSORBANCE

RESULT
From graph,
For the given sample the manganese concentration = __________mg/L.

DETERMINTION OF SULPHATES
(TURBIDIMETRIC METHOD)
AIM
To determine the volume of sulphates in given water sample with the stipulation as
per IS: 3025(part 24) –Reaffirmed 2003.

INTRODUCTION
Sulphates is widely distributed in nature and may be present in natural waters in
concentration ranging from few hundred to several thousand mg/L. Sulphates occur naturally
in numerous minerals, including barite, epsomite and gypsum. These dissolved minerals
contribute to the mineral content of drinking-waters. Acid Mine Drainage (AMD) may

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contribute large amounts of sulphates through pyrite oxidation. Sulfate is the second most
abundant anion in seawater. Its high concentration owes to the high to moderate solubility of
the salts that it forms with the major cations in seawater, namely, Na, Mg2+, and Ca2+.

PRINCIPLE
The turbid metric method of measuring sulphates is based upon the fact that barium
sulphates tends to precipitate in colloidal form of uniform size. And that this tendency is
enhanced in presence of a sodium chloride, hydrochloric acid and glycerol.
SO42- + BaCl2→BaSO4
The absorbance of the barium sulphates formed is measured by a spectrophotometer at 420
nm and the sulphates ion concentration is determined by comparison of the reading with a
standard curve.

APPARATUS REQUIRED
1) UV-Visible spectrometer
2) Sample tubes
3) Standard flask
4) Beaker
5) Spatula
6) Measuring cylinder
7) Wash bottle
8) Tissue paper

CHEMICALS REQUIRED
1) Isopropyl alcohol
2) Glycerol
3) Concentrated hydrochloric acid
4) Sodium chloride
5) Barium chloride
6) Sodium sulphate
7) Distilled water

PROCEDURE
Preparation of reagents
Conditioning reagents

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 1) Measure exactly 25 ml glycerol and pour it to a dry clean beaker.
2) Then, measure 15 ml of concentrated hydrochloric acid adds it to the same beaker.
3) To the same beaker, add exactly 50 ml of 95 % isopropyl alcohol and mix well.
4) Accurate weigh 37.5 g sodium chloride and dissolve it in distilled water.
5) Then mix all the contents and make up the final volume to 250 ml using distilled
water.

Standard sulphate solution

1) Weigh accurately 1.479 g anhydrous sodium sulphate and dissolve it in distilled
water.
2) Take 1000ml standard measuring flask and place a funnel over it.
3) Transfer it to the 1000 ml standard flask and make up to 1000 ml using distilled
water.
(1 ml = 1.0mg SO42- )

Preparation of blank, standards, and sample for testing

1) Take six 50 ml glass stoppered standard flask (four for standards, one for sample and
one for the blank).
2) Add 10 ml of the standard sulphate solution to the first standard flask, 20 ml to the
second, 30 ml to the third and 40 ml to the fourth.
3) To the fifth flask add 20 ml of the sample water.
4) The sixth standard flask is for the blank; to this standard flask add distilled water
alone.
5) Add 5 ml of conditioning reagent to all standard flasks.
6) Then make up the volume to the 100 ml mark using distilled water.

CALCULATION
Table 1: Data for calibration curve
SL.NO CONCENTRATION ABSORBANCE

Page 43 of 66
 4

From graph,
For the given sample the manganese concentration = __________mg/L.

DETERMINATION OF FLUORIDE
(ALIZARIN VISUAL CALORIMETRIC METHOD)
AIM
To determine the concentration of fluoride in the given water sample.
PRINCIPLE
Fluoride may occur naturally in water or it may be added in controlled amounts. A
fluoride concentration of 1.0 mg/l in drinking water effectively reduces dental caries without
harmful effects on health. In alizarin visual calorimetric method zirconium ions react with
alizarin to give red colour described as red lake. The colour intensity produced is proportional
to amount of zirconium present. When fluoride is present, it takes zirconium from the

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complex and hence the colour intensity is reduced. This colour intensity is proportional to the
fluoride concentration.
(Zirconium-Alizarin complex) + F- (Zr F6)2- + Alizarin (Yellow)
Then by usual calorimetric method or using a photometer the amount of fluoride in a
water sample can be determined
The bleaching action of fluoride is slow and therefore comparisons are made one hour
after mixing the lake and fluoride. In addition the colour intensity is temperature dependent.
Therefore, in this determination time and temperature are carefully maintained constant in
constructing the absorbance concentration calibration curve. Chloride, if present in water,
interferes with estimation. It must be eliminated by the addition of sodium arsenite solution.
APPARATUS
Calourimeter, Nesslers tubes and water bath
REAGENTS
1) Stock fluoride solution
Dissolve 221 mg of NaF and dilute to one litre 1ml=100mg of F-
2) Standard fluoride solution
Dilute 1000 ml stock fluoride solution to one litre 1ml = 10 mg of F-
3) Ziroconyl – Alizarin Reagent
Dissolve 300 mg Ziroconyl – Alizarin octahydrate ZerOCl 2.8H2O in 50 ml of
distilled water. Dissolve 70mg Alizarin solution into zircon solution with mixing. The
solution clears on standing for minutes.

4) Mixed acid solution

Dilute 101 ml conc. HCl to 400 ml. Add carefully 33.3 ml conc.H 2SO4 to 400
ml water. After cooling mix the two acids.
5) Acid Ziroconyl – Alizarin Reagent
To the clear zirconyl alizarin reagent add the mixed acid solution and make
upto one litre. The solution changes in colour from red to yellow within one hour.
Now it is ready for use. Store away from the direct sunlight to extent the stability to
the reagent upto 6 months.
6) Sodium Arsenite Solution
Dissolve 5g sodium arsenite and dilute to one litre (Poisonous)

Page 45 of 66
PROCEDURE
1. Sample Pre-treatment
If sample contains residual chlorine remove it by adding one- drop arsenite
solution for every 1.0 mg of residual chlorine
2. Preparing Standards
Prepare a series of standards by diluting different volumes of the standard
fluoride solution. (One ml=10 μg) to 100 CC in a Nessler tube. Choose the standards
so that at least there is one with a lower and one with higher fluoride concentration
than the sample. An interval of 50 μg per litre is usually sufficient i.e 0.5 ml
difference between the standards.
3. Colour Development
To 100 ml clear sample and the standards add 5ml acid zirconyl alizarin
reagent. Mix thoroughly and compare the colour after one hour. Plot a calibration
curve for standard fluoride solution finally calculates the concentration of fluoride in
the sample from calibration curve and express in mg/l.

RESULT
Amount of fluoride present in
1. Given Sample = _______________ mg/L.
2. Tap water = _______________ mg/L.

DETERMINATION OF IRON
(1, 10 – PHENANTHROLINE METHOD)

AIM
To determine the amount of iron present in the given sample
PRINCIPLE
In filtered sampled of surface waters iron concentration seldom reach 1mg/L. Some
ground waters and acid surface drainage contain considerably more iron. In water samples
iron may occur in true solution, in a colloidal state, in inorganic or organic complexes or in a
relatively suspended particle. In 1,10 phenanthroline method the ferric form of iron and

Page 46 of 66
hyroxlane. Later phenanthroline is added at pH between 3.2 and 3.3 to form soluble chelated
complex of orange red colour with iron. The intensity of colour developed is proportional to
the concentration of ferrous iron.
APPARATUS
Calorimeter, Nessler tubes, Acid washed glassware.
REAGENTS
1. Concentrate Hydrochloric acid
2. Hydroxylamine hydrochloride
Dissolve 10g hydroxylamine hydrochloride (NH2OHHCl) in 100ml water.
3. Ammonium Acetate Buffer
Dissolve 250g of ammonium acetate in 150ml distilled water. Add 140ml
acetic acid.
4. Phenanthroline solution
Dissolve 100mg 1,10 phenanthroline monohydrate in 100ml distilled water by
stirring and heating to 80 °C.
5. Stock iron solution
Dissolve 7.021g of Ferrous Ammonium Sulphate in one litre of distilled water
(or 0.7021g of Ferrous Ammonium Sulphate in 100ml of distilled water) which is
1000ppm solution (i.e. 1ml = 1mg Fe). From this various standards (1ppm to
100ppm) can be prepared.

PROCEDURE
Preparation of Calibration Curve
1. Take 10ml of 1000ppm solution and dissolve it in distilled water and make upto
100ml. This will be equal to 100ppm.
2. Take 10ml of the 100ppm solution and dissolve it in distilled water and make upto
100ml. This will be equal to 10ppm solution.
3. Similarly prepare 20ppm to 100ppm standards.
4. Take 50ml of each of the standards and add 2ml of hydrochloric acid 1ml of hydroxyl
amine hydrochloride 8 – 10ml of ammonium acetate buffer and 3 – 4ml of 1, 10
phenanthroline solution.

Page 47 of 66
 5. The colour intensity of orange red complex developed for each of the standards is
measured in terms of absorbance value at wavelength of 510nm.
6. A linear line is obtained by plotting concentration on the X axis and absorbance on
the Y axis.

Preparation of Water sample and Blank

1. Take 100ml of water sample is a clean conical flask and add 2ml of HCl and 1ml of
hydroxyl amine hydrochloride solution.
2. The above solution is heated to boiling and cooled to room temperature. Thus by
heating the ferric form of the iron is converted to ferrous form.
3. Now add 3-4ml of 1, 10 phenanthroline solutions and 8-10ml of ammonium acetate
buffer to the cooled samples to get the orange red complex.
4. The colour thus developed is measured in the calorimeter to get the absorbance value.
5. A reference or Blank is measured in the same manner as the water sample.

Determination of unknown concentration of water sample

The absorbance value of the blank is set in the colorimeter. Then the absorbance value of
the water sample is interpolated in the calibration in the calibration curve and the
corresponding concentration of ferrous iron in the water sample is measured.

RESULT
The amount of Ferrous iron present in the water sample = _______________ mg/L.

DETERMINATION OF CHEMICAL OXYGEN DEMAND

AIM
To determine chemical oxygen demand in the given water sample with the stipulations as per
IS: 3025 (Part 58) - Reaffirmed 2006.

INTRODUCTION
Before performing this experiment, few questions may arise to the learners:
 What is meant by chemical oxygen demand?
 Why do we need to determine COD?

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  What are the methods available to measure COD?
 Is it measured in water or wastewater?
 Whether is it mandatory to determine COD as per our codal provision?

The chemical oxygen demand (COD) test is commonly used to indirectly measure the
amount of organic compounds in water. Most applications of COD determine the amount of
organic pollutants found in surface water (e.g. lakes and rivers), making COD a useful
measure of water quality. It is expressed in milligrams per liter (mg/L), which indicates the
mass of oxygen consumed per liter of solution.
COD is the measurement of the amount of oxygen in water consumed for chemical oxidation
of pollutants.
COD determines the quantity of oxygen required to oxidize the organic matter in water or
waste water sample, under specific conditions of oxidizing agent, temperature, and time.
This method covers the determination of COD in ground and surface waters, domestic and
industrial wastewaters. The applicable range is 3-900 mg/L.

PRINCIPLE
The organic matter present in sample gets oxidized completely by potassium dichromate
(K2Cr2O7) in the presence of sulphuric acid (H 2SO4), silver sulphate (AgSO4) and mercury
sulphate (HgSO4) to produce CO2 and H2O. The sample is refluxed with a known amount of
potassium dichromate (K2Cr2O7) in the sulphuric acid medium and the excess potassium
dichromate (K2Cr2O7) is determined by titration against ferrous ammonium sulphate, using
ferroin as an indicator. The dichromate consumed by the sample is equivalent to the amount
of O2 required to oxidize the organic matter.

APPARATUS REQUIRED
1. COD Digester
2. Burette & Burette stand
3. COD Vials with stand
4. 250 mL conical flask (Erlenmeyer Flask)
5. Pipettes
6. Pipette bulb
7. Tissue papers
8. Wash Bottle

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CHEMICALS REQUIRED
1. Potassium dichromate
2. Sulfuric acid
3. Ferrous ammonium sulphate
4. Silver sulphate
5. Mercury sulphate
6. Ferroin indicator
7. Organic free distilled water

PREPARATION OF REAGENTS
a) Standard Potassium Dichromate Reagent - Digestion Solution
Weigh accurately 4.913 g of potassium dichromate, previously dried at 103ºC for 2 - 4 hours
and transfer it to a beaker.
Weigh exactly 33.3g of mercuric sulphate and add to the same beaker.
Measure accurately 167 mL of concentrated sulphuric acid using clean dry measuring
cylinder and transfer it to the beaker. Dissolve the contents and cool to room temperature. (If
not dissolved keep it over night).
Take 1000 mL standard measuring flask and place a funnel over it.
Carefully transfer the contents to the 1000 mL standard flask and make up to 1000 mL using
distilled water.
This is the standard potassium dichromate solution to be used for digestion.

b) Sulphuric Acid Reagent - Catalyst Solution

Weigh accurately 5.5 g silver sulphate crystals to a dry clean 1000 mL beaker. To this
carefully add about 500 mL of concentrated sulphuric acid and allow standing for 24 hours
(so that the silver sulphate crystals dissolve completely).

c) Standard Ferrous Ammonium Sulphate solution

Weigh accurately 39.2g of ferrous ammonium sulphate crystals and dissolve it in distilled
water.
Take 1000 mL standard measuring flask and place a funnel over it.
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Carefully transfer the contents to the 1000 mL standard flask and make up to 1000 mL mark
using distilled water.

TESTING OF SAMPLE
1. Take three COD vials with stopper (two for the sample and one for the blank).
2. Add 2.5 mL of the sample to each of the two COD vials and the remaining COD vial
is for blank; to this COD vial add distilled water.
3. Add 1.5 mL of potassium dichromate reagent - digestion solution to each of the three
COD vials.
4. Add 3.5 mL of sulphuric acid reagent - catalyst solution in the same manner.
5. CAUTION: COD vials are hot now.
6. Cap tubes tightly. Switch on the COD Digester and fix the temperature at 150º C and
set the time at 2 hours.
7. Place the COD vials into a block digester at 150°C and heat for two hours.
8. The digester automatically switches off. Then remove the vials and allow it to cool to
the room temperature.
9. Meanwhile, get ready with the burette for the titration.
10. Fill the burette with the ferrous ammonium sulphate solution, adjust to zero and fix
the burette to the stand.
11. Transfer the contents of the blank vial to conical flask.
12. Add few drops of ferroin indicator. The solution becomes bluish green in colour.
13. Titrate it with the ferrous ammonium sulphate taken in the burette.
14. End point of the titration is the appearance of the reddish brown colour.
15. Note down the volume of ferrous ammonium sulphate solution added for the blank (A) is
-----mL.
16. Transfer the contents of the sample vial to conical flask.
17. Add few drops of ferroin indicator. The solution becomes green in colour.
18. Titrate it with the ferrous ammonium sulphate taken in the burette.
19. End point of the titration is the appearance of the reddish brown colour.
20. Note down the volume of ferrous ammonium sulphate solution added for the sample (B) is -----
mL.

OBSERVATION AND CALCULATION

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For determining the Chemical Oxygen Demand in the given water sample, the readings
should be tabulated.
Burette Solution : Ferrous Ammonium Sulphate
Pipette Solution : Sample
Indicator : Ferroin Indicator
End point : Appearance of reddish brown color

TABLE
Sl No. Sample Volume of Burette Reading (mL) Volume of 0.1 N
Sample (mL) Initial Final FAS (mL)

Volume of Ferrous Ammonium sulphate for blank (A) = ----- mL

Volume of Ferrous Ammonium sulphate for Sample (B) = ----- mL
Normality of Ferrous Ammonium sulphate N = 0.1 N
Volume of Sample V = ----- mL

¿
Chemical Oxygen Demand = ( A−B)× N ×8 × 1000¿ Volume ofSample

RESULT
COD of the given sample = _______________ mg/L.

BIOLOGICAL PARAMETERS
DETERMINATION OF BIOLOGICAL OXYGEN DEMAND

AIM
To determine biochemical oxygen demand in the given water sample with the stipulations as
per IS: 3025 (Part 44) - Reaffirmed 2003.

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INTRODUCTION
The biochemical oxygen demand determination is a chemical procedure for determining the
amount of dissolved oxygen needed by aerobic organisms in a water body to break the
organic materials present in the given water sample at certain temperature over a specific
period of time.
BOD of water or polluted water is the amount of oxygen required for the biological
decomposition of dissolved organic matter to occur under standard condition at a
standardized time and temperature. Usually, the time is taken as 5 days and the temperature is
20°C.
The test measures the molecular oxygen utilized during a specified incubation period for the
biochemical degradation of organic material (carbonaceous demand) and the oxygen used to
oxidize inorganic material such as sulfides and ferrous ion. It also may measure the amount
of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand).

PRINCIPLE
The sample is filled in an airtight bottle and incubated at specific temperature for 5 days. The
dissolved oxygen (DO) content of the sample is determined before and after five days of
incubation at 20°C and the BOD is calculated from the difference between initial and final
DO.
The initial DO is determined shortly after the dilution is made; all oxygen uptakes occurring
after this measurement is included in the BOD measurement.

APPARATUS REQUIRED
1. BOD Incubator
2. Burette & Burette stand
3. 300 mL glass stopper BOD bottles
4. 500 mL conical flask
5. Pipettes with elongated tips
6. Pipette bulb
7. 250 mL graduated cylinders
8. Wash bottle

CHEMICALS REQUIRED
1. Calcium Chloride
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2. Magnesium Sulphate
3. Ferric Chloride
4. Di Potassium Hydrogen Phosphate
5. Potassium Di Hydrogen Phosphate
6. Di sodium hydrogen phosphate
7. Ammonium Chloride
8. Manganous sulphate
9. Potassium hydroxide
10. Potassium iodide
11. Sodium azide
12. Concentrated sulfuric acid
13. Starch indicator
14. Sodium thiosulphate
15. Distilled or deionized

PROCEDURE
For testing the given sample, first the reagents are required to be prepared.
PREPARATION OF REAGENT
a) Manganous Sulphate Solution
Dissolve Manganese Sulphate
→ 480g of MnSO4.4H2O (or)
→ 400g of MnSO4.2H2O (or)
→ 364 g of MnSO4 .H2O
In freshly boiled and cooled distilled water, filter the solution and make up to 1000 mL (One
litre). In this experiment, we are using Manganese sulphate Mono hydrate.

Take 364g MnSO4.H2O of and transfer it to the beaker. To dissolve the content, place it in
the magnetic stirrer
Note: The solution should not give blue color by addition of acidified potassium iodide
solution and starch.
b) Alkaline Iodide Sodium Azide Solution
To prepare this reagent we are going to mix three different chemicals
Dissolve either
→ 500 g of Sodium Hydroxide (or)
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 → 700 g of Potassium Hydroxide
→ 135 g of Sodium Iodide (or)
→ 150 g of Potassium Iodide
To prepare this reagent, take 700 g of potassium hydroxide and add 150 g of potassium
iodide and dissolve it in freshly boiled and cooled water, and make up to 1000 mL (One
litre).
Dissolve 10 g of Sodium Azide in 40 mL of distilled water and add this with constant
stirring to the cool alkaline iodide solution prepared.
c) Sodium Thiosulphate stock solution
Weigh approximately 25 g of sodium thiosulphate and dissolve it in boiled distilled water
and make up to 1000 mL. Add 1 g of sodium hydroxide to preserve it.
d) Starch Indicator
Weigh approximately 2 g of starch and dissolve in 100 mL of hot distilled water
e) Sulphuric Acid
In case if you are going to preserve the starch indicator add 0.2 g of salicyclic acid as
preservative.
f) Calcium Chloride solution
Weigh accurately 27.5 g of anhydrous calcium chloride and dissolve it in distilled water.
Take 100 mL standard measuring flask and place a funnel over it.
Transfer it to the 100 mL standard flask and make up to 100 mL using distilled water.
g) Magnesium Sulphate solution
Weigh accurately 22.5 g of magnesium sulphate and dissolve it in distilled water.
Take 100 mL standard measuring flask and place a funnel over it.
Transfer it to the 100 mL standard flask and make up to 100 mL using distilled water.
h) Ferric Chloride solution
Weigh accurately 0.15 g ferric chloride and dissolve it in distilled water.
Take 100 mL standard measuring flask and place a funnel over it.
Transfer it to the 100 mL standard flask and make up to 100 mL using distilled water.
i) Phosphate buffer solution
Weigh accurately 8.5g of Potassium Di Hydrogen Phosphate (KH2PO4) and dissolve it in
distilled water.
Then add exactly 21.75 g of Di Potassium Hydrogen Phosphate (K2HPO4) and dissolve it.
To the same beaker 33.4 g of Di sodium hydrogen phosphate (Na2HPO4 7H2O), is weighed
and added.
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Finally to the beaker containing all the salts, add accurately 1.7 g of Ammonium Chloride
(NH4Cl) and dissolve it.
Take 1000 mL standard measuring flask and place a funnel over it.
Transfer it to the 1000 mL standard flask and make up to 1000 mL using distilled water.
The pH should be 7.2 without further adjustment.
j) Dilution Water
High quality organic free water must be used for dilution purposes.
The required volume of water (five litres of organic free distilled water) is aerated with a
supply of clean compressed air for at least 12 hours. Allow it to stabilize by incubating it at
20ºC for at least 4 hours.
For the test we have taken five litres of organic free aerated distilled water, hence add 5mL
each of the nutrients.
 Add 5mL calcium chloride solution
 Add 5mL magnesium sulphate solution
 Add 5mL ferric chloride solution and
 Add 5mL phosphate buffer solution

This is the standard dilution water. Prepare dilution water 3 to 5 days before initiating BOD
test to ensure that the BOD of the dilution water is less than 0.2 mg/L.

TESTING OF SAMPLE
1. Take four 300 mL glass stoppered BOD bottles (two for the sample and two for the
blank).
2. Add 10 mL of the sample to each of the two BOD bottles and the fill the remaining
quantity with the dilution water. i.e., we have diluted the sample 30 times.
3. The remaining two BOD bottles are for blank, to these bottles add dilution water
alone.
4. After the addition immediately place the glass stopper over the BOD bottles and note
down the numbers of the bottle for identification.
5. Now preserve one blank solution bottle and one sample solution bottle in a BOD
incubator at 20ºC for five days.
6. The other two bottles (one blank and one sample) needs to be analysed immediately.
7. Avoid any kind of bubbling and trapping of air bubbles. Remember – no bubbles!

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8. Add 2mL of manganese sulfate to the BOD bottle by inserting the calibrated pipette
just below the surface of the liquid.
9. Add 2 mL of alkali-iodide-azide reagent in the same manner.
10. (The pipette should be dipped inside the sample while adding the above two reagents.
If the reagent is added above the sample surface, you will introduce oxygen into the
sample.)
11. Allow it to settle for sufficient time in order to react completely with oxygen.
12. When this floc has settled to the bottom, shake the contents thoroughly by turning it
upside down.
13. Add 2 mL of concentrated sulfuric acid via a pipette held just above the surface of the
sample.
14. Carefully stopper and invert several times to dissolve the floc.
15. Titration needs to be started immediately after the transfer of the contents to
Erlenmeyer flask.
16. Rinse the burette with sodium thiosulphate and then fill it with sodium thiosulphate.
Fix the burette to the stand.
17. Measure out 203 mL of the solution from the bottle and transfer to an Erlenmeyer
flask.
18. Titrate the solution with standard sodium thiosulphate solution until the yellow color
of liberated Iodine is almost faded out. (Pale yellow color)
19. Add 1 mL of starch solution.
20. and continue the titration until the blue color disappears to colourless.
21. Note down the volume of sodium thiosulphate solution added , which gives the
22. D.O. in mg/L. Repeat the titration for concordant values.
23. After five days, take out the bottles from the BOD incubator and analyse the sample
and the blank for DO.
24. Add 2mL of manganese sulfate to the BOD bottle by inserting the calibrated pipette
just below the surface of the liquid.
25. Add 2 mL of alkali-iodide-azide reagent in the same manner.
26. If oxygen is present, a brownish-orange cloud of precipitate or floc will appear.
27. Allow it to settle for sufficient time in order to react completely with oxygen.
28. When this floc has settled to the bottom, shake the contents thoroughly by turning it
upside down.

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 29. Add 2 mL of concentrated sulfuric acid via a pipette held just above the surface of the
sample.
30. Carefully stopper and invert several times to dissolve the floc.
31. Titration needs to be started immediately after the transfer of the contents to
Erlenmeyer flask.
32. Rinse the burette with sodium thiosulphate and then fill it with sodium thiosulphate.
Fix the burette to the stand.
33. Measure out 203 mL of the solution from the bottle and transfer to an Erlenmeyer
flask.
34. Titrate the solution with standard sodium thiosulphate solution until the yellow color
of liberated Iodine is almost faded out. (Pale yellow color)
35. Add 1 mL of starch solution and continue the titration until the blue color disappears
to colourless.
36. Note down the volume of sodium thiosulphate solution added, which gives the D.O.
in mg/L. Repeat the titration for concordant values.

OBSERVATION AND CALCULATION

For determining the Biochemical Oxygen Demand in the given water sample, the readings
should be tabulated.
Trial Day Volume Burette Reading Volume of Dissolved
No. of Sample Initial Final Titrant (mL) Oxygen (mg/L)
(ml)

Calculation:
Initial DO of the diluted sample, D0 = mL
DO at the end of 5 days for the diluted sample, D5 = mL
Blank correction = C0 - C5, BC = mL
Initial DO of the blank, C0 = mL
DO at the end of 5 days for the blank, C5 = mL

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Biochemical Oxygen Demand = {D0− D5 − BC} x Volume of the diluted sample
Volume of sample taken

OBSERVATION AND CALCULATION

1. 0th day DO of blank = ___________ mg/l
2. 0th day DO of sample = ___________ mg/l
3. 5th day DO of sample = ___________ mg/l

RESULT
Biochemical Oxygen Demand of the given sample = ______________ mg/l

MICROBIOLOGY EXAMINATIONS

BACTERIOLOGICAL EXAMINATION OF WATER SAMPLE

 Preparation of Nutrient medium
 Straining Procedure for Microbial Specimens

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  Standard Plate Count Test (Dilution technique & plating)
 Test for coliforms

1. DILUTION TECHNIQUE & PLATING

AIM
To determine the microbial population in a given sample.

PRINCIPLE

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 When sampling the given item quantitative bacteriological analysis, dilutions are often
used to determine the no. of organism in a given volume. If dilution are not carried out
growth till the so numerous that cannot be counted.

REQUIREMENTS
1) Water blanks containing 18ml of sterib buffered water.
2) Petri dish
3) Sample to be examined
4) Pipettes
5) Nutrient Agar
6) Glass tubes
7) Non absorbent cotton

PROCEDURE

1) Set up the sample bottles & test tubes containing sterile buffered water.
2) Underneath arrange the petri dish in two lines, corresponding to sample bottles &
test tubes.
3) Add 2ml of sample to a water blank containing 18ml of sterile buffer water and mix
it; thoroughly by rotating tubes bath the palms of the hand.
4) Transfer one ml to a petri dish and label it as 1:100
5) Transfer 2ml of 1:10 dilution to another water blank 18ml and mix thoroughly &
label it as 1:100
6) Transfer 1ml from the 1:100 dilution to a petri dish and label it as 1:100
7) Transfer 2ml of dilution to another water blank & mix it thoroughly & label it as
1:1000
8) Add 1ml to a petri dish & label it as 1:1000
9) Transfer 2ml of 1:1000 to another water blank & mix it thoroughly & label it as
1:10000
10) Like mix prepare serial dilution according to sample characters
11) Flame the neck of nutrient agar tube & pore 10ml to a each petri dish including the
control
12) Spread the agar, evenly over the bottom of the petri dish by rotating the petri dish.
Take care that agar should not splash over the petri dish.
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 13) Inaculate the petri dish at 37 0 c for 24hrs and count the colonies by using a colony
counter.

BUFFER WATER PREPARATION

1) 34 g of potassium dihydrogen phosphate in 500ml of distilled water. Adjust the pH

to 7.4 & dilute to 1 litre (first solution)
2) 50 g of MgSo4 in 1litre of distilled water
BUFFER WATER

Take 1.25 ml first solution & 5ml from the second solution , dilute to 1 litre.

OBSERVATION

Pettri dishs are taken after incubation period and no. of colonies are counted &
tabulated.

2. TEST FOR COLIFORMS

1. PRESUMPTIVE TEST

AIM
To find the pressure of total coliforms in a given sample.

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PRINCIPLE:
Coliforms are used as indicator organisms. The presence of coliforms reveals the
extent of fecal contamination in a given sample.

REQUIREMENTS
1) Sample bottle
2) test tubes
3) pipettes
4) Dehydrated Mac-conkey broph
5) Fermentation tubes
6) cotton (non-absorbant)

PROCEDURE

1) Prepare double strength Mac-conkey broth medium by using the standard quality
water and doubling the ingredient
2) 10 ml of the solution is put in a test tube in each
3) take care while inserting the fermentation tube so that there should not be any air
bubble
4) dilute to double strength medium with equal quantity of water
5) 10ml of this single medium is put in a test tube along with a fermentation
6) auto clave the medium at 121ºC for 15 minutes and 15 lps pressure
7) Collect the water sample in a sample bottle with dew precautions to avoid any on
site contamination
8) Put 10ml of the sample to each of the test tube containing double strength medium
sterile pipette
9) Put 1 ml of the sample to each of the test tubes containing normal strengths with
usual precautions
10) Put 0.1 ml of the sample to each test tube containing normal strengths with usual
precautions
11) Keep 5 test tube for each dilution (10 ml, 1 ml, 0.1ml)
12) incubate all the test tubes are the rate of 37ºC for 24 hours
13) observe the change of colour in the test tube in the test tube
14) If gas production and colour change do not appear keep it for another 24 hours
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PREPARATION OF MAC-CONKEY BROTH

Dehydrated media - 40g/l

Peptone - 20 g/l
Lactose - 10g bile salt/l
NaCl - 5 g/l
Bromocresol purple - 1% solution
pH - 7.4

3. CONFIRMATIVE TEST
[Total coliform & Fecal coliform]

AIM
To confirm the presence of coliform which have given the +ve presumptive test.
Requirements:-
1. presumptive +ve tubes
2. brilliant lactose bile broth

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 3. fermentation tubes
4. inaculation needle
5. test tubes
6. cotton

PROCEDURE
1. Prepare brilliant green lactose bile broth medium.
2. Distribute it to a test tube at the rate of 5ml /tube and put a fermentation in an inverted
form to each tube.
3. Autoclave it at 1200 c for 15 min and 15 lps pressure
4. Cool the medium and transfer one or two loopful of sample from the +ve presumptive
tube to BGLP medium under sterile atmosphere.
5. Inaculate another BGLP medium with the same manner as described above.
6. Incubate one set of BGLP medium,@ 370c for 48hrs.This is for fecal coliforms.
7. Look for colour change and gas collection in the fermentation tube after 24 hrs and 48
hrs.

PREPARATION OF BGLP MEDIUM

1. dehydrated medium- 4gm/lt
2. peptone- 10 gm/lt
3. Lactose- 10 gm/lt
4. bile salt-20gm/lt
5. brilliant green- 1% solution ie,[1.33ml]
6. pH- 7.4

4. CONFIRMATIVE TEST FOR E.Coli

AIM
To determine the pressure of E.coli in a given sample which gave +ve presumptive result
in the Mac-Conkey broth medium.

REQUIREMENTS
1) test tubes

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 2) peptones
3) sodium cholide
4) inaculation needle
5) +ve presumptive test tubes
6) indole reagent
7) xylene

PROCEDURE
1) peptone water is prepared in sufficient quantity and poured into the test tube at the rate
of 5 ml/tube
2) Autoclave the medium at 1210c for 15min at 15 lps pressure
3) Cool the medium and transfer one or two loopful of tube from the positive presumptive
to the peptone water under sterile water.
4) Incubate the test tube at 440c at 24hrs.
5) After 24 hrs add 1 ml of xylene and few drops of indole reagent to each test tube. Shake
it well and leave it for 10 min.
6) Observe the colour and record it if red ring develops. It indicates presence of E.Coli.

PREPARATION OF PEPTONE WATER

Peptone-10gm
Nacl-5 gm
Distilled water –1 lt
PREPARATION OF INDOLE REAGENT
Dissolve 5 gm paradimethyl aminobenzaldehyde in 75 ml isopropyl alcohol and add
25 ml of concentrated HCL.

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