Dharmsinh Desai University, Nadiad

Department of Chemical engineering

prepared by:
Prof. J.M.Jivani
Introduction

Raw Water

Surface Sub-surface
Sources Sources

Ponds, Lakes, Streams, Rivers,

Springs, Infiltration wells,
Storage reservoirs, Oceans(not
Well and Tube-wells
used for water supply)
Wastewater: is simply that part of the water supply to the community or
to the industry which has been used for different purposes and has been
mixed with solids either suspended or dissolved.

Wastewater is 99.9% water and 0.1% solids. The main task in treating
the wastewater is simply to remove most or all of this 0.1% of solids.

3
 Physical, chemicals and biological properties
of wastewater

Characteristic Sources
Physical properties: Domestic and industrial wastes, natural decay of organic
Color materials
Odor Decomposing wastewater, industrial wastes.
Solids Domestic water supply, domestic and industrial wastes, soil
erosion, inflow infiltration
Temperature Domestic and industrial wastes
Chemical constituents:
Organic:
Carbohydrates Domestic, commercial, and industrial wastes
Fats, oils, and grease Domestic, commercial, and industrial wastes
Pesticides Agricultural wastes
Phenols Industrial wastes
Proteins Domestic, commercial, and industrial wastes
Priority pollutants Domestic, commercial, and industrial wastes
 Surfactants Domestic, commercial, and industrialwastes

Volatile organic compounds Domestic, commercial, and industrialwastes

Other Natural decay of organic materials

Inorganic: Domestic wastes, domestic water supply, groundwater infiltration

Aikalinity
Chlorides Domestic wastes, domestic water supply, groundwater infiltration

Heavy metals Industrial wastes

Nitrogen Domestic and agricultural wastes

PH Domestic, commercial, and industrial wastes

Phosphorus Domestic, commercial, and industrial wastes naturalrunoff

Priority polluter Sulfur Domestic water supply; doestic, commercial. And industrial wastes

Gases: Decomposition of domestic wastes

Hydrogen sulfide
Methane Decomposition of domestic wastes

Oxygen Domestic water supply , surface- water infiltration

Biological constituents: Open watercourses and treatment plants

Animals
Plants Open watercourses and treatment plants

Eubacteria Domestic wastes, surface water infiltration, treatment plants .

Archaebacteria Domestic wastes, surface-water infiltration, treatment plants

Viruses Domestic wastes

Characteristics of Water

Physical Chemical Bacteriological

• Turbidity • Total Dissolved Solid • Indicator Organism

• Colour • pH Value • Total Coliform
• Taste • Hardness • Fecal Coliform
• Odour • Alkalinity
• Temperature • Fluoride
• Density • Organics
(BOD,COD,TOC)
• Metals
• Nutrients
Characteristics of Water

Physical Characteristics
Characteristics of Water
A) Physical Characteristics
1) Suspended Solids:
 Total solids = Suspended +Dissolved
Sources: inorganic , organic particles and immiscible liquids
 Inorganic Solids- Clay, silt and soil constituents
 Organic materials- Plant fibres and biological solids(algal cells,
bacteria, etc.)
 Immiscible liquids- Oil, grease etc.
Impacts: may be objectionable in water for several reasons.
 Aesthetically displeasing and provides adsorption sites for
chemical and biological agent.
 Characteristics of Water
 Organic solids may be degraded biologically, resulting in
objectionable by-products.
 Biologically active(live) suspended solids may include disease
causing organism.
 Desirable amount = 500 ppm
Higher upto 1000 ppm
Measurement:
 SS can be removed and measure by filtration
 Filter the water, drying residue to a constant weight at 104°C(± 1°)
 Determine the mass of the residue retained on the filter paper
(mg/L).
Characteristics of Water
2) Turbidity:
 Is a measure of the extent to which light is either absorbed or
scattered by suspended material in water.
 Not a direct quantitative measurement of suspended solids
because of absorption and scattering are influenced by both
size and surface characteristics of ss materials.
Sources:
 Clay, silt, rock and metal oxides from the soil, vegetable fibres
and microorganism also contribute to turbidity.
 Household and industrial wastewater
 Desirable limit = 5 NTU
 Permissible limit = 5-10 NTU
NTU- Nephlometric Turbidity Unit
Characteristics of Water
Impacts:
 Aesthetically displeasing opaqueness or milky
 Colloidal materials (clay, silt, etc) provide adsorption sites for
chemicals that may be harmful or cause undesirable taste and
odor.
Measurement:
 Turbidimeters- Photometrically
 Nephlometers -NTU(Nephlometric Turbidity Unit)
 Jackson turbidimeter- JTU
 Principle: NTU - based on scattering of light
JTU – based on absorption of light
1 JTU = 1 mg SiO2/ 1 lit of Distilled Water
Characteristics of Water
Characteristics of Water
3) Colour:
In nature water is colored by foreign substances.
Dissolved Solid- True Color
Suspended Solid- Apparent Color
Sources:
Industrial waste from textile, pulp and paper, food processing,
chemical production etc.
Iron Oxide- reddish water
Manganese Oxide- Brown or Blackish water
Impacts:
Not acceptable
Carcinogens are produced after combination of chlorine with
color producing organics.
Characteristics of Water
Measurement:
TCU (True Color Unit)
1 TCU = color produced by 1mg/L of platinum cobalt
1, 2,3,_,_,_,_,_mg platinum cobalt in 1 lit of distilled water
1,2,3,_,_,_,_,_ ppm platinum cobalt solution
Apparatus: Tintometer
Permissible Range- < 10
4) Taste and Odor:
Substance that produce an odor in water will almost impart a
taste as well.
But many substance produce taste but no odor.
Characteristics of Water
Sources:
Minerals, metals and salt from the soil, end products from biological
reactions and constituents of waste water.
Dissolved gases – H2S, CH4, CO2, etc.
Impacts:
Aesthetically displeasing
Odors produced by organic substances, some of those substances
may be carcinogenic.
TON (Threshold odour number) = (A+B)/A
A = the volume of sample
and B = the volume of odor-free water.
Always A+B = 200 mL
Equipment Used to analyze odors include:
Butanol wheel
scentometer
Characteristics of Water
Q1. Find the TON when odor is first detected in a flask containing
50 mL of sample water.
• TON = 200ml/Sample Volume,ml
= 200ml/ 50 ml = 4
5) Temperature:
Affect chemical reactions that occurs in natural water system
Affects the biological activity of bacteria
Temp - Biological Activity -
Also affects the solubility of gases
Viscosity with decreasing in temperature
Max. density of water occurs at 4°C.
Also effect the solubility of gases in water
Characteristics of Water

6) Density:
( Assignment question )
Characteristics of Water

Chemical Characteristics
Characteristics of Water
1. Total Dissolved Solid:
 Determined by evaporating a sample of water and weighing
the dry residue.
Sources:
Inorganic and organics
Inorganic- Minerals, metals and gases
Organic- Decay Products of vegetation, organic chemicals, organic
gases etc.
Impacts:
 Undesirable in water
 May also produce color, taste and odor.
 Some chemicals may be toxic and carcinogenic.
Characteristics of Water
Desirable limit = 500 ppm
Permissible limit = 2000ppm
EC also give approximate analysis of TDS.
EC- Conductivity Meter- Wheatstone bridge principle at
25°C

2) pH Value:
pH = - log[H+]
[H+] = 10-pH ( mole/lit)
Other units- mg/L or mg/L as CaCO3
< 7 – acidic
>7 – Basic
= 7 - Neutral
Characteristics of Water
Pure water is a balanced combination of positively charged ion
and negatively charged ions.
Sources:
Alkalinity is caused by the presence of bicarbonates of Ca and
Mg or
By the carbonates or hydroxides of Na, K, Ca & Mg
Acidity is caused by the presence of minerals acids, CO2,
sulfates of iron and aluminum.
Measurement:
Potentiometer(pH meter) - electrical potential exerted by
hydrogen ion and indicating their concentration.
Characteristics of Water
Impacts:
Acidic water – Corrosion
Alkaline water- Sediment deposits, Difficulties in chlorination,
certain psychological effects on human system etc.
Desirable range = 6.5 to 8.5

3) Hardness of water:
Which prevent the formation of sufficient leather or foam,
when hard water mixed with soap.
Salts of Ca & Mg
 Hardness of Water

Temporarily Hardness Permanent Hardness

Carbonates and bicarbonates of Ca

& Mg
Removed- Simple Boiling or by
adding lime
Ca(HCO3)2 =CaCO3 + CO2 + H2O
Mg(HCO3)2 =Mg(OH)2 + 2CO2
Sulfates, chlorides and
nitrates of Ca & Mg
It require special
treatment for softening
Characteristics of Water
Sources:
Multivalent metallic ions in natural water are Ca & Mg
May include iron and manganese in reduced state (Fe 2+,
Mn2+), strontium(Sr2+) and aluminum (Al3+)
Hardness = Ions of ( Ca + Mg)
Impacts:
Sodium soap + multivalent metallic cations  Precipitate
2NaCO2C17H33 + cations2+ Cations2+(CO2C17H33 ) 2(ppt) + 2Na+
Ppt adhere to surface of tubes, sinks and may stain clothing,
dishes and other items
Remain in the pores of skin, so that skin may feel rough
Lead to greater soap consumption, scaling of boilers causing
corrosion
Characteristics of Water
Desirable Mg conc. < 50 mg/L , also exceed this amount
Ca Hardness = 75 mg/L, No public health effect
Measurement:
Titration- EDTA (0.01M) as titrant and EBT as an indicator
Color changes Red  Blue
Total Hardness(mg/L as CaCO3) = Ca Hardness + Mg Hardness

Total Hardness = (Carbonate + Non- Carbonate)Hardness

Carbonate Hardness = Total Hardness Or Alkalinity whichever
is lesser
Characteristics of Water
If TH > Alk
Then Carbonate Hardness(CHs) = Alkalinity
Non-Carbonate Hardness(NCHs) = TH - CHs
If TH < Alk
Carbonate Hardness(CHs) = TH
Non-Carbonate Hardness(NCHs)= Nil

Ques.1. Determine the Carbonate and Non-Carbonate Hardness

of a water having a total alkalinity of 200 mg/L as CaCO3, and
120 mg/L of Ca++ and 60 mg/L of Mg++ as ions.
TH = (120/20 + 60/12) x 50 = 550 mg/L as CaCO3
CHs = Alk = 200 mg/L as CaCO3
NCHs = TH- CHs = 550- 200 = 350 mg/L as CaCO3
Characteristics of Water
Classification of water on the basis of Hardness

Water Category Hardness Conc. (mg/L as CaCO3)

Soft < 50

Moderately Hard 50 – 150

Hard 150 -300

Very Hard > 300

Characteristics of Water
4) Alkalinity:
Is a measure of the ability of water to neutralize acids
Quantity of anions in water to neutralize H+ ions.
Sources:
Dissolution of minerals in soil and atmosphere
Main- CO3-- , HCO3-, OH-
Other- HSiO3-, HS-, NH3, H2BO3- etc.
 Little negative alk is also caused by H+
PO43- - from detergents, fertilizers and insecticides from
agricultural land
H2S, NH3 - Microbial decomposition of organic material
Total alk = (CO3-- + HCO3- + OH- - H+ ) mg/L as CaCO3
Characteristics of Water
Impacts:
In large quantity- impart bitter taste to water
Desirable =200 mg/L as CaCO3
Permissible = 600 mg/L as CaCO3
Measurements:
 Titrating sample with acid
 Titrant - N/50 H2SO4, Indicator- Bromocresol green(5-6 drops)
 End Point: Green  Yellow
1 mL of acid will neutralize – 1mg of alk as CaCO3
N1V1 = N2V2
Alkalinity (mg/L as CaCO3) = mL volume of acid neutralized x 10
Characteristics of Water
Ques: If the pH of water is 10. Determine total alkalinity of a water
containing 90 mg/L of CO 32- ions and 61 mg/L of HCO3- ions.
Solution:
Total alkalinity = (CO3-- + HCO3- + OH- - H+ ) mg/L as CaCO3
CO 32- as CaCO3 = 90 x (50/30) = 150
HCO3- as CaCO3 = 61 x (50/61) = 50
H+ = 10-10 moles/L =10-7 mg/L = 5 x 10-6 mg/L as CaCO3
OH- = 5 mg/L as CaCO3
Total alkalinity = 150 + 50 + 5 - 5 x 10-6
= 205 mg/L as CaCO3
 Characteristics of Water
Ques: In a water treatment plant, the pH values of incoming and
outgoing water are 7.2 and 8.4 respectively. Assuming a linear
variation of pH with time, determine the avg. pH value of water.
Solution: pH = - log[H+]
(pH)1 = 7.2 = - log[H1+]
(pH)2 = 8.4 = - log[H2+]
[H1+] = 10 -7.2 , [H2+] = 10-8.4
Avg. value of [Havg+] = [H1+] + [H2+] /2
= 10-7.477
Avg. value of pH = - log[Havg+]
pH (avg.) = 7.477
Characteristics of Water
5)Fluoride:
• Appreciable quantities – surface water
• Few geographical regions – ground water
• In large quantities - toxic for human and animals
• Small quantities – can be beneficial
conc. 1.0 mg/L – help to prevent dental cavities in children
• During formation of teeth  F- + teeth enamel  Harder and
stronger teeth
Conc. > 2.0 mg/L  Discoloration of teeth
Excessive dosage of F-  Bone fluorosis and other skeletal
abnormalities
Desirable Range: 1.0 mg/L
Permissible range: 1.5 mg/L
Characteristics of Water
6) Organics:
Starch, fats, protein, alcohols, acids, ether, aldehydes, etc
Sources:
• Many organics are soluble in water
• Comes from natural resources or from human activities
Dissolved organics:
i. Biodegradable organics
ii. Non- Biodegradable organics
Biodegradable organics: Consists of organics that can be utilized
for food by naturally occurring micro-organism.
BOD: Biochemical oxygen demand (biologically active)
Std BOD: 5 day BOD at 20°C ( BOD5 )
Measurements of organic matter:-
Many parameters have been used to measure the concentration of organic
matter in wastewater. The following are the most common used methods:

Biochemical oxygen demand (BOD).

BOD5 is the oxygen equivalent of organic matter. It is determined by measuring

the dissolved oxygen used by microorganisms during the biochemical oxidation
of organic matter in 5 days at 20oC

Chemical oxygen demand (COD)

It is the oxygen equivalent of organic matter. It is determined by measuring

the dissolved oxygen used during the chemical oxidation of organic matter in
3 hours.
Total organic carbon (TOC)

This method measures the organic carbon existing in the wastewater

by injecting a sample of the WW in special device in which the carbon
is oxidized to carbon dioxide then carbon dioxide is measured and
used to quantify the amount of organic matter in the WW. This method
is only used for small concentration of organic matter.

Theoretical oxygen (ThOD)

If the chemical formula of the organic matter existing in the WW is

known the ThOD may be computed as the amount of oxygen needed
to oxidize the organic carbon to carbon dioxide and a other end
products.

35
Characteristics of Water
Measurement:
• Incubating diluted sample for 5 days at 20°C
• Std BOD bottles – 300 mL

Dilution Factor = Vol. of diluted sample/ Vol. of undiluted sample

Non- Biodegradable organics:
• Not degrade under biological degradation
• Includes: lignic acid, cellulose, phenols, etc.
COD(Chemical oxygen demand)= Biologically (active+ inactive)
Measurement: COD analyzer, may also with TOC analysis
Characteristics of Water
Ques: Determine BOD5 of a waste water if three dilution are
prepared to cover this range. First the sample is placed in the
std BOD bottle and is then diluted to 300mL with oxygen and
organic free water.
Given data:
Waste DOi Dof DO Dilution BOD5
water (mg/L) (mg/L) consumed Factor (mg/L)
(mL) (mg/L)
5 9.2 6.9
10 9.1 4.4
20 8.9 1.5
Characteristics of Water
Solution:
Waste DOi Dof DO Dilution BOD5
water (mg/L) (mg/L) consumed Factor (mg/L)
(mL) (mg/L)
5 9.2 6.9 2.3

10 9.1 4.4 4.7

20 8.9 1.5 7.4

Characteristics of Water
Solution:
Waste DOi Dof DO Dilution BOD5
water (mg/L) (mg/L) consumed Factor (mg/L)
(mL) (mg/L)
5 9.2 6.9 2.3 60

10 9.1 4.4 4.7 30

20 8.9 1.5 7.4 15

Characteristics of Water
Solution:
Waste DOi Dof DO Dilution BOD5
water (mg/L) (mg/L) consumed Factor (mg/L)
(mL) (mg/L)
5 9.2 6.9 2.3 60 138

10 9.1 4.4 4.7 30 141

20 8.9 1.5 7.4 15 111

Biological Oxygen Demand (BOD):
The following are the theoretical equations used to calculate the BOD.
The Figure shown is used to describe the change of BOD with time. From the
figure the following correlations are derived:

L 0  or (BOD ultimate ) or UBOD.

Yt = BODt (BODexerted).
Lt = L0 e-kt (BOD remain).
BODt = L0 - Lt = L0 – L0e-kt = L 0(1-e-kt)
BOD5 = L0 (1-e-k5)
K = 0.23d-1 usually, k T = k20  T-20 ,  = 1.047 or as given
Example
Determine the 1-day BOD and ultimate BOD for
a wastewater whose 5- day 20 °C BOD is 200
mg/L. The reaction constant K= 0.23d-1 what would
have been the 5-day BOD if it had been conducted at 25°C?

Solution:-
• BODt = UBOD – BODr = UBOD (1-e-kt) =L 0(1-e-kt)
200 = L0 (1-e-0.23x5)
L0 = 293 mg/L (this is UBOD)
• Determine the 1-day BOD:-
BODt = L0 (l-e-kt)
BOD1 = 293 (l-e-0.23x1) = 60.1 mg/L
• Determine the 5-day BOD at 25C:-
KT = K20 (1.047)T-20  K25 = 0.23 (1.047)25-20
BOD5 = L0 (l-e –kt ) = 293 (l-e-0.29x5) = 224 mg/L

42
Characteristics of Water
7) Metals:
• All metals are soluble to some extent in water
• Excessive amount – health hazards
Sources:
• Dissolution from natural deposits
• Discharge of domestic, industrial and agricultural waste water
Measurement:
• By atomic absorption spectrophotometery
Type:
i. Non-toxic metals
ii. Toxic metals
Characteristics of Water
i. Non-toxic metals: ( Ca, Mg, Na, Fe, Mn, Cu, and Zn)
Na:
• Found in natural water in earth’s crust
• Highly reactive with other elements
• Na-salts are very soluble in water
• Excessive conc. – bitter taste in water , health hazard to
kidney patient and toxic to plant
• Also corrosive to metal surfaces
Fe & Mn:
• Quite frequently occurs together
• In natural conc. – no health hazard
• very small quantity – color problem(Fe=0.3, Mn=0.05 mg/L)
Characteristics of Water
• Energy source for some bacteria – growth may produce taste &
odor problem
• In natural water Fe- associated with chloride (FeCl2),
bicarbonate [Fe(HCO3)2] , sulfate (FeSO4 ) and exist in reduced
state
• In presence of O2
Fe+2 + O2  Fe+3  Fe(OH)3 ↓ (insoluble)
• Mn+2 & Mn+4 associated with Cl, SO4 & NO3 are soluble
• Mn+3 & Mn+5 are insoluble
Impacts:
• Non-toxic metals are found in small quantities in natural water
& cause taste problem long before toxic levels were reached.
Characteristics of Water
Cu & Zn:
• In small quantities may be toxic
ii. toxic metals:
• Arsenic, barium, cadmium, chromium, lead, mercury etc.
• Toxic metals are harmful to human and other organism in
small quantities
• May be dissolved in water

8) Nutrients:
• Elements essential to the growth and reproduction of plants,
animals and aquatic species
• Nutrients required by aquatic species are C, N and P
 Characteristics of Water
Carbon:
• CO2 – from atmosphere
• Alkalinity and decay product of organic matter all supply carbon to
the aquatic system
Nitrogen:
• Primary component of earth’s atmosphere and extremely stable
• N2 gas + O2 (under high energy)  Nox
• Constituent of protein, chlorophyll and many other biological
compounds
• After the death of plants
Complex org compound (bacterial decomposition)  Amino acids
(reduced)  NH3
Characteristics of Water
• If O2 is present
NH3  NO2-  NO3- (Nitrate)
Sources:
• Animal wastes, chemicals and wastewater discharge
• NH3 ↔ aqueous ionic NH4+
• Nitrate Poisoning(Blue baby disease) in infant, animal and
human
Phosphorus:
• Appears as PO43- in aquatic environment
• Other forms- orthophosphate, condensed phosphate(pyro,
meta and poly phospate)
• May be soluble
• Constituent of plant and animal tissue
Characteristics of Water
• Non-toxic
• No direct health hazard to human and other organism
Characteristics of Water

Biological Characteristics
Biological Characteristics:-

The environmental engineer must have considerable knowledge of the

biological of waste water because it is a very important characteristics
factor in wastewater treatment.

The Engineer should know:-

1. The principal groups of microorganisms found in wastewater.

2. The pathogenic organisms.
3. Indicator organisms (indicate the – presence of pathogens).
4. The methods used to amount the microorganisms.
5. The methods to evaluate the toxicity of treated wastewater
Main groups of Microorganisms:-
The main microorganisms of concern in
wastewater treatment are Bacteria,
Fungi, Algae, Protozoa, Viruses, and pathogenic
microorganisms groups.
Bacteria:-
Types: Spheroid, rod curved rod, spiral,
filamentous. Some important bacteria:-
Pseudomonas:- reduce NO3 to N2, So it is very importantin
biological nitrate removal in treatment works.
Zoogloea:- helps through its slime production in
the formation of flocs in the aeration tanks.
Sphaerotilus natuns:Causes sludge bulking in
the aeration tanks. Bdellovibrio: destroy
pathogens in biological treatment.
Acinetobacter: Store large amounts of
phosphate under aerobic conditions and
release it under an – anaerobic condition so,
they are useful in phosphate removal.
Nitrosomonas: transform NH4 into NO2-
Nitrobacter: transform NO2- to NO 3-
Coliform bacteria:- The most common type is E-Coli or
Echerichia Coli, (indicator for the presence of pathogens).
E-Coli is measured in (No/100mL)

Fungi:
• Important in decomposing organic matter to simple forms.
Algae:
• Cause eutrophication phenomena. (negative effect)
• Useful in oxidation ponds. (positive effect)
• Cause taste and problems when decayed. (negative effect)
Protozoa:
•Feed on bacteria so they help in the purification of treatedwaste water.
•Some of them are pathogenic.
Characteristics of Water
Bacteria:
• Single cell organism possessing no defined nucleous
• Having no green material(chlorophyll) to produce food
• Reproduced by binary fission
• Small length (1-4 micron)
• Can’t be seen by naked eye and Examined with microscope
• Virus are much smaller and can’t be seen by microscope and
their presence can be detected by observing their reaction to
different stains or culture
Characteristics of Water
Shapes:
1. Spherical shape ( single, pair, chain & irregular branch)
2. Straight rod
3. Spiral
4. curved
Characteristics of Water
Type:
• Non-pathogenic Bacteria(Non-pathogens)
Harmless, can be beneficial to human, animals and crops (eg.
Lactobacillus used for curd formation)
• Pathogenic Bacteria (Pathogens)
Capable of infecting or transmitting disease to human &
animals
Can be transported by natural water system
Water bone pathogens- bacteria, virus, protozoa and parasitic
worms
Water bone diseases – cholera, typhoid, infectious, hepatitis
etc
Characteristics of Water
Depending upon the oxygen necessities of the bacteria, they may
be classified as follows:
I. Aerobic bacteria
II. Anaerobic bacteria
III. Facultative bacteria
Measurement:
Routine test are generally conducted only to detect and count the
presence of indicator organism
Indicator organism:
• Presence and absence in water indicates the nature and extent
of contaminants or presence and absence of coliform pollution
Characteristics of Water
• Most widely used indicator organism are coliform group (total &
fecal) for drinking water
• Fecal coliform is the subgroup of total coliform
Methods for counting coliform:
i. Membrane filter technique or membrane filter test
ii. Multiple tube fermentation technique

Membrane filter technique or membrane filter test

• Filter the water sample through sterilize membrane of special
design (pore size 5-10 micrometer, porosity 80%), on which the
bacteria will be retained
Characteristics of Water
• Membrane is placed upon a plate saturated with a suitable
nutrient medium and incubated at 35°C
• Bacteria are able to grow upon nutrient medium and will produce
visible colonies
• These colonies can be counted and each colony representing one
bacterium in original sample
• This incubation process is known as culturing through the culture
medium
• Medium & temperature of incubation depends upon the type of
bacteria
• Total coliform count, M-Endo broth or LES Endo Agar are used
with incubation at 35°C for 20-22 hrs
Characteristics of Water
• For fecal coliform count, medium to be used is M-FC broth and
incubation at 44.5°C for 22 hrs

Multiple tube fermentation technique

• In this test small tubes called Duhram tubes placed in test tubes in
inverted position
• A broth containing Lactose (10 mL) is placed in a series of test
tubes
• 5 test tubes are inoculated(mixed) with 10 mL of water sample in
each tube
• Another 5 test tubes are inoculated(mixed) with 1 mL and again
another 5 test tubes are inoculated(mixed) with 0.1 mL
Characteristics of Water
• These tubes are finally incubated at 35°C for 48 hrs
• Formation of gas in small tubes will give a positive presumptive
test of the presence of coliform organism
• If no gas is evolved, means it will show a negative test ( absence of
coliform)
• For fecal test a portion from the fermentation tubes is transferred
to a tube containing green bile broth
• This is incubated for 48 hrs at 42°C, and if gas is formed- positive
confirmatory test (presence of fecal coliform)
• If no gas is formed negative test
• Count the no. of positive test tubes and then read the count from
MPN index table
Characteristics of Water
• Suppose 5-3-1 test tubes are positive
Then, from table the coliform count is 110 MPN index/100 mL
MPN- Most Probable Number
30 January 2021 63