Sewage Water Analysis

1

ABBREVIATIONS AND SYMBOLS

g gram

μL micro liter

ºC degree Celsius

% percentage

ppm parts per millions

mg/L milligrams per liter

hrs. Hours

mins Minutes

mM millimolar

N Normality

M Molarity

Tur Turbiduty

D.W. distilled water

B.R. Burette reading

ST sample taken

SS Suspended solids

TDS total dissolved solids

COD chemical oxygen demand

2

BOD biochemical oxygen demand

DO dissolved oxygen

TC total coliform

FC faecal coliform

E.coli. Escherichia coli

MPN most probable number

FAS ferrous ammonium sulphate

O.D. Optical Density

nm nanometer

STD standard

3

CONTENT

Chapter Page No.

1. Introduction 5 – 17

2. Materials & Methods 18 – 34

4. Results & Discussion 35 – 47

References 48 – 49

List of Tables

Table:1 Physical & Microbial parameter of Site 1 from

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Table:14 Physical & Microbial parameter of Site 14 from final outlet

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CHAPTER: 1 INTRODUCTION

1.1 ENVIRONMENT

"Environment is surrounding atmosphere/ condition for

existence" OR "Environment is an essential natural process

or an outcome of occurrence"

It means, our Environment is our surrounding which includes

living and non-living things around us. The non-living

components of environment are land, water and air. The living

components are germs, plants, animals and people. Due to our

selfish purposes we create disturbances in it and thus, as a result

imbalance in nature can be seen from few decades which is

increased with the increase of time. The main cause of imbalance

is pollution, which may be produced due to different

anthropogenic activities. Here we are going to discuss on

pollution, types of pollution and its effects on plants and animals

with the assessment of aqueous sample by different parameters.

1.2 POLLUTION

Pollution is the introduction of contaminants into a natural

environment that causes instability, disorder, harm or discomfort to

the ecosystem, physical systems or living organisms. Pollution can

take the form of chemical substances or energy, such as noise, heat

or light. Pollutants, the components of pollution, can be either

foreign substances/energies or naturally occurring contaminants.

1.2.1 Environmental pollution is any discharge of material or

energy into water, land, or air that causes or may cause acute

6

(short-term) or chronic (long-term) detriment to the Earth's

ecological balance or lowers the quality of life. Or any

unfavourable change or degeneration in the environment is

known as Environmental Pollution.

1.2.2 Pollutant: A pollutant is a waste material that pollutes air,

water or soil. Three factors determine the severity of a pollutant:

its chemical nature, the concentration and the persistence.

1.3 WATER is one of the most important elements on earth.

Every living being needs water for its survival. Without water,

plants, animals, microbes– everything will perish. Population

growth - coupled with industrialization and urbanization has

resulted in an increasing demand for water thus leading to water

crisis and serious consequences on the environment. The

requirement of fresh water for industrial use will increase from

30 BCM (Billion Cubic Meters) to 120 BCM by 2025 AD. A

rapid industrialization has lead to the industrial effluents and

sewage, resulting in water pollution which leads to water crisis in

India and all over the world.

1.3.1 WATER POLLUTION, by the discharge

of wastewater from the commercial and industrial

waste (intentionally or through spills) into surface waters;

discharges of untreated domestic sewage, and chemical

contaminants, such as chlorine, from treated sewage; release of

waste and contaminants into surface runoff flowing to surface

waters (including urban runoff and agricultural runoff, which

may contain chemical fertilizers and pesticides); waste disposal

7

and leaching into groundwater; eutrophication and littering. This

process ranges from simple addition of dissolved or suspended

solids to discharge of the most insidious and persistent toxic

pollutants (such as pesticides, heavy metals, and nondegradable,

bio accumulative, chemical compounds).

Fig.1 WATER POLLUTION

1.4 HARMFUL EFFECTS OF WATER POLLUTION ON

PLANTS, ANIMALS AND HUMANS

1.4.1 EFFECTS ON PLANTS:

� May disrupt photosynthesis in aquatic plants and thus

affecting ecosystems that depend on these plants.

8

� Terrestrial and aquatic plants may absorb pollutants from

water (as their main nutrient source) and pass them up the food

chain to consumer animals and humans.

� Plants may be killed by too much sodium chloride (ordinary

slat) in water.

� Plants may be killed by mud from construction sites as well

as bits of wood and leaves, clay and other similar materials.

� Plants may be killed by herbicides in water; herbicides are

chemicals which are most harmful to plants.

1.4.2 EFFECTS ON ANIMALS

� Nutrient pollution (nitrogen, phosphates etc) causes

overgrowth of toxic algae eaten by other aquatic animals, and

may cause death

� Oil pollution (as part of chemical contamination) can

negatively affect development of marine organisms, increase

susceptibility to disease and affect reproductive processes; can

also cause gastrointestinal irritation, liver and kidney damage,

and damage to the nervous system.

� Mercury in water can cause abnormal behavior, slower

growth and development, reduced reproduction, and death

Too much sodium chloride (ordinary salt) in water may kill

animals.

9

1.4.3 EFFECTS ON HUMANS

We know that pollution causes not only physical disabilities but

also psychological and behavioural disorders in people. The

following pollution effects on humans have been reported:

a) Waterborne diseases caused by polluted drinking water

� Typhoid

� Amoebiasis

� Giardiasis

� Ascariasis

b) Waterborne diseases caused by polluted beach water:

� Rashes, ear ache, pink eye

� Respiratory infections

� Hepatitis,encephalitis,gastroenteritis,diarrhoea,

vomiting,and stomach ache

c) Conditions related to water polluted by chemicals (such as

pesticides, hydrocarbon, organic pollutants, heavy metals etc):

� Cancer, incl. prostate cancer and non-Hodgkin’s lymphoma

� Hormonal problems that can disrupt reproductive and

developmental processes

� Damage to the nervous system

� Liver and kidney damage

� Damage to the DNA

� Damage to people may be caused by vegetable crops grown

or washed with polluted water .

� The effects of water pollution are not always immediate.

They are not always seen at the point of contamination. They are

10

sometimes never known by the person responsible for the

pollution. However, water pollution has a huge impact on our

lives. With knowledge, consideration and preparation, water

pollution can be decreased. It doesn't take much effort -- just a

little thought

1.5 SEWAGE AND WASTEWATER

� Domestic households, industrial and agricultural practices

produce wastewater that can cause pollution of many lakes and

rivers..

� Waste Water is any water that has been adversely affected

in quality by anthropogenic influence. It is , basically the flow of

used water from a community.

� The nature of wastewater includes physical, chemical, and

biological characteristics which depend on the water usage in the

community, the industrial and commercial contributions,

weather, and infiltration/inflow. It is 99.94 percent water by

weight (Water Pollution Control Federation 1980). The

remaining 0.06 percent is material dissolved or suspended in the

water.

� Sewage is the term used for wastewater that often contains

faeces, urine and laundry waste. It also includes domestic,

municipal, or industrial liquid waste products disposed of,

usually via a pipe or sewer or similar structure, sometimes in

acesspool emptier

� There are billions of people on Earth, so treating sewage is

a big priority.

11

� Sewage disposal is a major problem in developing countries

as many people in these areas don’t have access to sanitary

conditions and clean water.

� Untreated sewage water in such areas can contaminate the

environment and cause diseases such as diarrhoea.

� Sewage in developed countries is carried away from the

home quickly and hygienically through sewage pipes.

� Sewage is treated in water treatment plants and the waste is

often disposed into the sea.

� Sewage is mainly biodegradable and most of it is broken

down in the environment.

� In developed countries, sewage often causes problems when

people flush chemical and pharmaceutical substances down the

toilet. When people are ill, sewage often carries harmful viruses

and bacteria into the environment causing health problems.

Wastewater or sewage can come from:

� Human waste (faces, used toilet paper or wipes, urine, or

other bodily fluids), also known as black water, usually from

lavatories

� Septic tank discharge

� Sewage treatment plant discharge

� Washing water (personal, clothes, floors, dishes, etc.), also

known as grey water or silage;

� Rainfall collected on roofs, yards, hard-standings, etc.

(generally clean with traces of oils and fuel)

� Groundwater infiltrated into sewage

12

� Surplus manufactured liquids from domestic sources

(drinks, cooking oil, pesticides, lubricating, paint, cleaning

liquids, etc.)

� Urban rainfall runoff from roads, car parks, roofs,

sidewalks, or pavements (contains oils, animal faces, litter, fuel

or rubber residues, metals from vehicle exhausts, etc.)

� Direct ingress of manmade liquids (illegal disposal of

pesticides, used oils, etc.)

� Highway drainage (oil, de-icing agents, rubber residues)

� Storm drains (almost anything, including cars, shopping

trolleys, trees, cattle, etc.)

� Black water (surface water contaminated by sewage)

� Industrial waste

� industrial site drainage (silt, sand, alkali, oil, chemical

residues)

� Industrial cooling waters

� Industrial process waters

1.5.2 WASTEWATER CONSTITUENTS

The composition of wastewater varies widely. The partial list is

as follows:

Water ( > 95%) which is often added during flushing to carry

waste down a drain.

� Pathogens such as bacteria (Salmonella and Vibrio cholera)

, viruses (Hepatitis and Norwalk) prions and parasitic worms

(Cryptosporidium and Schistomsoma)

� Non-pathogenic bacteria;

13

� Organic particles such as faeces, hairs, food, vomit, paper

fibres, plant material, humus, etc.;

� Soluble organic material such as urea, fruit sugars, soluble

proteins, drugs, pharmaceuticals, etc.;

� Soluble inorganic material such as ammonia, road-salt, sea-

salt, cyanide, hydrogen sulphide etc.

� Animals such as protozoa, insects, arthropods, small fish,

etc.;

� Macro-solids such as sanitary napkins, nappies/diapers,

needles, children's toys, dead animals or plants, etc.

� Gases such as hydrogen sulphide, carbon dioxide, methane,

etc.;

� Emulsions such as paints, adhesives, mayonnaise, hair

colorants, emulsified oils, etc.;

� Toxins such as pesticides, poisons, herbicides, etc.

� Pharmaceuticals and other hormones.

To treat the discharge water from building and colonies

effectively, we are engaged in offering sewage treatment plant

and effluent treatment plant. These sewage treatment plant and

effluent treatment plant are efficiently assisting in better

treatment of extra polluted water from various industries, housing

complexes, colonies. Our effluent & sewage treatment plants are

functions as per the BOD and COD load and within the

permissible limits. These sewage treatment plant and effluent

treatment plant are appreciated by the clients for safeguarding the

environment from severely polluted water.

14

Figure 2 shows a typical schematic of an example wastewater

treatment process providing primary and secondary treatment

using the activated sludge process. There are three commonly

used approaches namely trickling filter, activated sludge, and

oxidation ponds in the secondary treatment. It provides

biochemical oxygen demand (BOD) removal beyond what is

achievable by simple sedimentation (Spellman, 2003).

Fig. 2 Schematic diagram of an example wastewater

treatment process providing primary and secondary

treatment using activated sludge process

15

1.6 STEPS OF SEWAGE TREATMENT PLANT:

Wastewater treatment is a series of steps. Each of the steps can be

accomplished using one or more treatment processes or types of

equipment. The major categories of treatment steps are:

1. PRELIMINARY TREATMENT - Removes materials that

could damage plant equipment or would occupy treatment

capacity without being treated.

2. PRIMARY TREATMENT - Removes settle able and

floatable solids (may not be present in all treatment plants).

3. SECONDARY TREATMENT - Removes BOD and

dissolved and colloidal suspended organic matter by biological

action. Organics are converted to stable solids, carbon dioxide

and more organisms.

4. DISINFECTION - Removes microorganisms to eliminate or

reduce the possibility of disease when the flow is discharged.

1.6.1 PRELIMINARY-TREATMENT:

Pre-treatment removes materials that can be easily collected from

the raw waste water before they damage or clog the pumps and

skimmers of primary treatment clarifiers (trash, tree limbs,

leaves, etc.).

1.6.1.1 SCREENING

The influent sewage water is screened to remove all large objects

like cans, rags, sticks, plastic packets etc. carried in the sewage

stream.

This is most commonly done with an automated mechanically

raked bar screen in modern plants serving large populations,

16

whilst in smaller or less modern plants a manually cleaned screen

may be used.

1.6.1.2 GRIT REMOVAL

Pre-treatment may include a sand or grit channel or chamber

where the velocity of the incoming wastewater is adjusted to

allow the settlement of sand, grit, stones, and broken glass.

1.6.2 PRIMARY TREATMENT:

In the primary sedimentation stage, sewage flows through

large tanks, commonly called "primary clarifiers" or "primary

sedimentation tanks." The tanks are used to settle sludge while

grease and oils rise to the surface and are skimmed off. Primary

settling tanks are usually equipped with mechanically driven

scrapers that continually drive the collected sludge towards a

hopper in the base of the tank where it is pumped to sludge

treatment facilities. Grease and oil from the floating material can

sometimes be recovered for saponification.

The dimensions of the tank should be designed to effect

removal of a high percentage of the floatables and sludge. A

typical sedimentation tank may remove from 60 to 65 percent of

suspended solids, and from 30 to 35 percent of biochemical

oxygen demand (BOD) from the sewage.

1.6.3 SECONDARY TREATMENT:

Secondary treatment is designed to substantially degrade

the biological content of the sewage which is derived from

human waste, food waste, soaps and detergent. The majority of

municipal plants treat the settled sewage liquor using aerobic

biological processes. To be effective, the biota requires both

17

oxygen and food to live. The bacteria and protozoa consume

biodegradable soluble organic contaminants and bind much of

the less soluble fractions into flock.

Secondary treatment systems are classified as fixed-film or

suspended-growth systems.

- FIXED-FILM or attached growth systems include trickling

filters and rotating biological contactors, where the biomass

grows on media and the sewage passes over its surface.

- SUSPENDED-GROWTH systems include activated

sludge, where the biomass is mixed with the sewage and can be

operated in a smaller space than fixed-film systems that treat the

same amount of water

1.6.4 TERTIARY TREATMENT:

� The pollutants were not sufficiently removed in secondary

treatment;

� As solids, nitrogen, phosphorus, and other pollutants such as

color and metals.

� The purpose of tertiary treatment is to provide a final

treatment stage to raise the effluent quality before it is discharged

to the receiving environment (sea, river, lake, ground, etc.).

� Tertiary treatment are of 3 types:

- FILTRATION: Sand filtration removes much of the residual

suspended matter

- NUTRIENT REMOVAL: Wastewater may contain high

levels of the nutrients nitrogen and phosphorus. In certain ways

that can be toxic to fish.

18

- DISINFECTION: The purpose of disinfection in the

treatment of waste water is to substantially reduce the number of

microorganisms in the water to be discharged back into the

environment.The effectiveness of disinfection depends on the

quality of the water being treated (cloudiness, pH, etc.)

Chapter: 2 PRINCIPLES, MATERIAL AND

METHOD OF THE PARAMETERS TO BE

ASSESSED:

2.1.1 PHYSICAL PARAMETERS:

1. COLOR :

� The color of industrial waste is considered to be the color of

light transmitted by the water after removing the suspended

matter.

� Colour in the water may results from natural metallic ions

(Mg & Fe), humus and peat materials, planktons, weeds, and

industrial waste. Coloured industrial wastewater may require

colour removal before discharge into water courses.

� Colour- It means true colour, that is the colour of water

form which turbidity is removed.

� Apparent colour- Includes not only colour due to

substances in solution, but also that due to suspended matters.

� Apparent colour is determined on the original sample

without filtration or centrifugation.

� In some highly coloured industrial wastewater colour is

contributed principally by colloidal or suspended material.

19

� The colour of the wastewater typically depends upon the

different industrial processes. The measurement and removal of

colour is essential part as it is unfit for recycling without proper

treatment

� Methods used:-

� Visual comparison method

� Spectrophotometric-single wavelength method.

� Significance:

- Consumer acceptance decreases, hinders the normal

photosynthetic activities in natural water bodies.

� Method:

� Visual Comparison Method/Platinum Cobalt Method

� Principle:

� The term color is used here to mean “true color”, that is,

the color of water from which turbidity has been removed.

The term “apparent color” includes not only color due to

substances in solution but also that due to suspended matter.

� Collection of sample: -

� Collect the sample in the plastic carbouy.

� Preservation:-

� Ice.

� Apparatus :-

20

� Color comparator

� Color disks

� Glass rod

Fig.3 COLOR COMPARATOR AND COLOR DISKS

� Procedure:-

� Take sample in beaker and shake well.

� Check color in color comparator by filling the test tube

with well shake sample and compare with D/W kept left side of

the comparator.

� Compare the color by checking disk of apparatus.

� If necessary carry out the dilution of the sample and add

this dilution factor in reading.

� Unit of color is Hazen or platinum cobalt.

21

� Calculation: -

Color Hazen or Pt.Co. Scale = Reading × Dilution

� Interferences: -

� Turbidity – it is removed by filtration or by centrifugation.

� pH- color value increases as the pH of water is raised.

2. SOLIDS:

� Solids- “Matters that suspended or dissolved in the water or

waste water”.

� It may affect the quality of water and waste water.

� High DS in water indicate inferior palatability.

� Desirable limit for drinking water for dissolved solids -

500mg/l.

� Total solids- Material residue left in the vessel after

evaporation of sample & its subsequent drying in an oven at a

defined temperature.

� Total solids includes-

- Total dissolved solids

- Suspended solids

- Fixed solids

- Volatile solids- weight loss on ignition

- Settlable solids.

22

A. Total dissolved solids:

� Desirable limit for drinking water of dissolved solids –

Ranges from 20 to 1000mg/l

� Hardness increases with TDS

� Filter paper used are of two types,

- Whatman filter paper - 42 mesh size , 47mm ,2μ pore size

- GF/C - glass fiber filter paper, 2μ pore size, resist high

temperature, 47mm, made up of glass wool.

� Main contributors of TDS- SO4-, Alkalinity as CaCO3,

Hardness as Ca, Mg, Nitrate,

� Silicates, Borates, Phosphates, Na, K ,F.

� In TDS , 0.6 of alkalinity`s results is participated.

� Significance: Highly mineralized water is less acceptable

than water with a moderate mineral content for drinking and

household purposes.

� Method:

� Gravimetric / Filtration Method.

� Principle:

� A well-mixed sample is filtered through a standard glass

fiber filter, and the filtrate is evaporated to dryness in a weighed

dish and dried to constant weight at 180°C. The increase in dish

weight represents the total dissolved solids. This procedure may

be used for drying at other temperatures. The results may not

agree with the theoretical value for solids calculated from

23

chemical analysis of sample. Approximate methods for

correlating chemical analysis with dissolved solids are available.

The filtrate from the total suspended solids determination may be

used for determination of total dissolved solids.

� Collection of sample :


� Preservation:-

� Ice

� Apparatus:

� Vacuum Filtration Assembly

� Graduated 50ml Cylinder

� Beakers

� Glass microfiber filter Paper GF/C (47mm)

� Oven

� Balance

24

FIG.4 MEMBRANE FILTER ASSEMBLY

� Procedure:

� Pre weighed the beaker (A)

� Assemble the Vacuum Filteration Assembly and filter.

� Take a well mixed 50ml sample and filter through glass fiber

filter.

� Wash with three successive 10ml volumes of D/W, allowing

complete drainage between washings and continue suction for

about 3 min after filteration is complete.

� Transfer filtrate to a preweighed beaker.

� Dry the beaker at 180 ºC ± 2 for 24 hours.

� Cool the beaker in desiccators and weigh the beaker(B).

�

� Calculations: -

25

��

Where,

A =weight of empty beaker or evaporating dish (crucible)

B= weight of dried residue + beaker or evaporating dish

(crucible)

� Interference:-

� Highly mineralized water with the significant concentration

of Calcium, Magnesium, Chloride, and/or sulfate may be

hygroscopic and require prolonged drying, proper desiccation,

and rapid weighing.

� Sample high in bicarbonate require careful and possibly

prolonged drying at 180ºC to insure complete conversion of

bicarbonate to carbonate. Because excessive residue in the beaker

may form a water-trapping crust.

B. TOTAL SUSPENDED SOLIDS:

� Significance:

� Suspended Solids impairs feeding ability through reduced

vision or interference with collecting mechanisms of filter

feeders in an aquatic ecosystem.

� Method:

� Gravimetric /Filtration Method

26

� Principle :-

� A well-mixed sample is filtered through a weighed standard

glass-fiber filter and the residue retained on the filter is dried to a

constant weight at 103 to 105°C. The increase in weight of the

filter represents the total suspended solids. If the suspended

material clogs the filter and prolongs filtration, it may be

necessary to increase the diameter of the filter or decrease the

sample volume. To obtain an estimate of total suspended solids,

calculate the difference between total dissolved solids and total

solids.

� Collection of sample:-


� Preservation:-

� Ice

� Apparatus:

� Vacuum Filteration Assembly

� Graduated 50ml Cylinder

� Glass microfiber filter Paper GF/C (47mm)

� Oven

� Balance

27

Fig.5 GLASS MICROFIBER FILTER PAPER GF/C

(47MM) AND FILTER PAPER ON WHICH SAMPLE IS

RUNNED.

28

� Procedure:

� Pre weighed the Glass microfiber filter Paper GF/C (47mm)

� Assemble the Vacuum Filteration Assembly and filter paper.

� Take a well mixed 50ml sample and filter through glass fiber

filter.

� Wash with three successive 10ml volumes of D/W, allowing

complete drainage between washings and continue suction for

about 3 min after filteration is complete.

� Carefully remove the filter paper from Vacuum Filteration

Assembly and transfer it to petridish.

� Dry the Glass microfiber filter Paper GF/C at 103 to 105 ºC for

atleast 1 hour.

� Weigh the Glass microfiber filter Paper GF/C after cooling (B).

� Calculation: -

��

Where,

A = weight of empty filter paper

B = weight of filter + dried residue

� Interference:-

� Large Floating Particle/Submerged agglomerates of

nonhomogeneous materials

29

2.1.3 BIOLOGICAL PARAMETERS:

1) Total COLIFORM and FECAL COLIFORM

� Another most common and widespread health risk

associated with drinking water is the bacterial contamination

caused either directly or indirectly by human or animal excreta.

� Assessment of indicator bacteria namely coliform bacteria

is a convenient way to evaluate potability and sanitary conditions

of water bodies.

� The bacterial species E.coli is a typical coliform bacteria

traditionally used as hygiene indicator bacteria, and methods for

their detection are essential for drinking water regulations all

over the world.

� Significance:

� These parameters give an idea about the contamination of

drinking water directly or indirectly by sewage or by human or

animal excreta. The contamination results into communicable

enteric disease. Presence of coliform bacteria in water supplies

indicates inadequate treatment or post treatment contamination.

E.coli is always present in the faceless of man. Animals & birds.

Tootle plate count or colony court is used to assess the general

bacterial contamination.

� Method :

� Most Probable Number

� Principle:

30

� It is statistical method based on the probability theory. In

this technique, the sample is serially diluted till the numbers of

organisms reach the point of extension. From each of these

dilutions several multiple tubes of a specific medium are

inoculated. Presence of organism is indicated by acid and gas in

the medium. Pattern of positive and negative test results are then

used to estimate the number of bacteria in the original sample.

Since the test gives the most probable number of organisms

present in the sample it is also known as MPN test.

� Collection of samples

� collect the sample in sterile glass bottle.

� Preservation:

� ice

� Apparatus:

� autoclave,

� Incubator,

� Gas burner,

� Nichrome wire loop

� ,Sterile pipettes and

� Sterile dilution water bottles.

� Reagents :

1. MacConkey’s Lactose Bile Broth (MLBB)

� Pepton-20.0 g

31

� Lactose-10.0 g

� Sodium Chloride-5.0 g

� Bile salts-3.0 to 5.0 g

� Neutral red-30.0 g

� Crystal violet-10.0 g

� Distilled water-1000 ml

� Agar-30.0 g

� pH-7.4

� Dissolve by heating; adjust pH to 7.4 and sterilize by

autoclaving. Above composition will make Single strength, But

double the concentration will make it Double strength medium

in 1000 ml distilled water.

2. Brilliant green lactose bile broth:

� Peptone-10 g

� Lactose- 10g

� Oxgall- 20 g

� Brilliant green- 0.0133 g

� Distilled water 1 litre

� pH should be 7.2 after sterilization and is then ready for use

� 1 MLBB tube having 50 ml double strength (2X)

medium.

� 5 MLBB tubes each having 10 ml double strength (2X)

medium.

� 11 MLBB tubes each having 5 ml single strength (X)

medium.

32

� Sterile 10 ml and 1 ml pipettes.

� Water sample to be tested.

3. Kovac’s reagent:

� P-dimethylaminobenzaldehyde-5 g

� Amyl alcohol- 75 ml

� Concentrated HCl - 25 ml

� Dissolve aldehyde in alcohol by gently warming in a

water bath.Cool and add with care.

� Procedure:

� Shake the water sample vigorously to ensure uniform

distribution of organisms.

� Dilute the sample (if necessary) by transferring 10 ml of

water sample by sterile pipette to 90 ml of sterile distilled water.

This makes 10-1

dilution. Make more dilutions if needed.

� With the sterile graduated pipettes inoculate the water

sample (diluted sample, if the dilution is done) as follows.

� 1 MLBB tube having 50 ml double strength (2X) medium

with 50 ml sample.

� 5 MLBB tube having 10ml double strength (2X) medium

with 10 ml sample each.

� 5 MLBB tube having 5ml single strength (X) medium with

1 ml sample each.

� 5 MLBB tube having 5ml single strength (X) medium with

0.1 ml sample each.

33

� One tube of MLBB having 5 ml (X) medium is left

uninoculated, which serves as control.

� Incubates all tubes at 37 0C for 24 hours.

� Examine tubes for acid and gas after 24 hours.

� If no tube shows acid and gas reincubate all tubes for

another 24 hours.

� At the end of the incubation period, record the number

of positive tubes in each of three sets (i.e. 10 ml, 1 ml and 0.1

ml), and interprete results as follows.

� Confirm test :

� Transfer the 1-2 loopful of culture from the presumptive

tube in each of two brilliant green broth each of 5ml fermentation

tube and incubate one tube at 37�C for 24 hr and other tubes at

44�C for 24 hr. the fermentation of gas in the tube after 24 hr.48

hr shows positive test.also incubate 1-2 loopful of culture in

peptone water tube(each of 5 ml) and incubate at 44�C for 24 hr.

� Interpretation:

� McCrady in 1918 computed tables (refer Apendix-1)

regarding the most probable number of organisms present in 100

ml of water, on the basis of various combination of positive and

negative results in the amounts used for tests. Number of

organisms per 100 ml is read from the McCrady’s table, and the

number is multiplied by the dilution factor (if any), to come to

the final number.

34

Fig.15 MOST PROBABLE NUMBER

� Calculation:

��

��

� Find out MPN / 100 ml by the help of MPN index.

� Tubes which are showing positive (gas formation) result at

37�C are coliform organism and positive at 44�C are fecal

coliform organisms.

� While addition of Covac’s reagent in peptone water tube

,the red color is developed, it indicates positive result.

� Interference :

� To avoid contamination, procedure should be done in sterile

condition.

35

Chapter: 3 RESULTS AND DISCUSSION

Table No. 1: Physical & Microbial parameter of Site 1 from final

outlet


outlet

Site

2

Microbial

parameterPhysical parameter

TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

2400 930 10 8.31 794 64

Site:

1

Microbial


TC FC Color

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

4300 2300 30 8.36 880 56

36


outlet

Site 3

Microbial parameter Physical parameter

TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

2500 950 20 8.04 986 94


outlet

Site 4


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

950 250

10 8.31 918 26

37

Table No.5: Physical & Microbial parameter of Site 5 from final

outlet

Site 5


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

2000 750 50 8.44 1382 52


outlet

Site:

6


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

1000 560 20 8.12 622 46

38


outlet

Site:

7


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

14000 600 50 7.08 556 120


outlet

Site:

8


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

930 250 10 7.89 1510 84

39


outlet

Site:

9


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

15000 2300 40 7.10 650 130

Table No.10: Physical & Microbial parameter of Site 10 from

final outlet

Site: 10

Microbial


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

930 150 40 7.10 650 130

40

Table No. 11: Physical & Microbial parameter of Site 11 from

final outlet

Site:

11


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

15000 4300 70 7.49 1138 4


final outlet

Site:

12


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

2800 1500 200 8.71 1596 70

41


final outlet

Site:

13


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

930 210 30 8.41 790 4


final outlet

Site:

14


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

750 430 30 8.28 968 36

42


final outlet

Site:

15


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

930 230 30 8.35 1004 18


final outlet

Site:

16


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

930 430 30 7.45 802 10

43


final outlet

Site:

17


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

7500 900 60 7.50 1004 12


final outlet

Site:

18


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

15000 4300 200 7.19 1166 749

44


final outlet

Site:

19


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

640 140 10 7.30 784 18


final outlet

Site:

20


TC FCColor

(Hazen)pH

TDS

(mg/l)

SS

(mg/l)

9300 4300 10 7.33 986 22

Comparative ana

Graph: 1 Compe

Graph: 2 Compe

0

50

100

150

200

250

Site1

site2

site3

site4

Ha

zen

0

2

4

6

8

10

site1

site2

site3

site4

site5

pH

alysis of different parameters

erative analysis of color from different

erative analysis of pH from different sit

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

location

Color

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

location

pH

45

site

te

Graph: 3 Compe

Graph: 4 Compe

0

500

1000

1500

2000

site1

site2

site3

site4

TD

Sm

g/L

0

100

200

300

400

500

600

700

800

site1

site2

site3

site4

SS

mg

/L

erative analysis of TDS from different s

erative analysis of SS from different sit

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

Location

TDS

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

Location

SS

46

site

te

Graph: 5 Comper

different site

Graph: 6 Compe

different site

0

2000

4000

6000

8000

10000

12000

14000

16000

site1

site2

site3

MP

N/

10

0m

l

0

1000

2000

3000

4000

5000

site1

site2

site3

site4

MP

N/

10

0m

l

rative analysis of total coliform from

erative analysis of total fecal coliform f

site4

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

Location

Total Coliform

site4

site5

site6

site7

site8

site9

site10

site11

site12

site13

site14

site15

site16

site17

site18

site19

site20

Location

Fecal Coliform

47

from

site20

site20

48

REFRENCES:

� Standard Methods of the Examination of Water and

Wastewater.

� ETD

� Shodhganga.

� Prescott, Harley, and Klein's Microbiology

� ASTM D1426 - 08 Standard Test Methods for Ammonia

Nitrogen In Water

� Johnson, D. L.; Ambrose, S. H.; Bassett, T. J.; Bowen, M.

L.; Crummey, D. E.; Isaacson, J. S.; Johnson, D. N.;

Lamb, P. et al (1997). "Meanings of Environmental

Terms". Journal of Environmental Quality 26 (3): 581–

589.doi:10.2134/jeq1997.00472425002600030002x

� "Pollution - Definition from the Merriam-Webster Online

Dictionary". Merriam-webster.com. 2010-08-13.

Retrieved 2010-08-26.

� Schueler, Thomas R. "Microbes and Urban Watersheds:

Concentrations, Sources, & Pathways."eprinted in The

Practice of Watershed Protection. 2000. Center for

Watershed Protection. Ellicott City, MD.

49

� United States Geological Survey (USGS). Denver, CO.

"Ground Water and Surface Water: A Single Resource."

USGS Circular 1139. 1998.

� Schueler, Thomas R. "Microbes and Urban Watersheds:

Concentrations, Sources, & Pathways." Reprinted in The

Practice of Watershed Protection. 2000. Center for

Watershed Protection. Ellicott City, MD.

� EPA. “Illness Related to Sewage in Water.” Accessed

February 20, 2009.

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