Microbiology of water

Bacteriological analysis of water

Potable or non-polluted water

Water which is free of pathogenic microorganisms and
chemical substances deleterious to health is referred to
as potable or non-polluted water.
The primary objectives for providing potable water are
to make water free from harmful microorganisms and
from undesirable or harmful chemicals.
There are methods to detect bacterial contamination of
water. The chief objective is to identify coliform
organisms as Escherichia coli. Their presence
indicates that water contains fecal pollution and is
unsafe for consumption.
Determination of water potability

Water generally gets contaminated with pathogenic

microorganisms through intestinal discharges of
humans and animals (i.e., faecal contamination) and
becomes an important source of water-borne diseases.
It is, therefore. important that the water must be
regularly analyzed to determine whether it is potable
or polluted with faecal matter.
Certain bacteria particularly Escherichia coli and
related organisms called coliforms, faecal
Streptococci (e.g., Streptococcus faecalis) and
Clostridium perfringens normally inhabit the large
intestine of humans and other animals and are frequent
present in their faces. Thus, the presence of any of
these microorganisms in water provides evidence of
its being faecally polluted.
 Bacteriological examination of water actually employs
three different techniques:
(1) total count,
(2) membrane filter method and
(3) multiple tube method.
Degree of Faecal Pollution of Water

The degree of faecal pollution of water is estimated by

colilitre or coli-index.
The colilitre is the smallest amount of water in which
one Escherichia coli is present.
The coli-index is the number of individuals of E.coli
found in one litre of water.
Water is considered potable if the colilitre is within
the limits of 300-500. It is considered to be of good
quality if the coli-index is 2-3.
To ensure the potability of water, i.e., whether the
water is potable or faecally polluted, generally two test
techniques are employed.
These techniques are : coliform-test-technique and
membrane-filter-technique.
Bacteriological techniques

Sterile bottle
Contamination is avoided
Tested promptly
Stored at 0 to 4C
i) Plate count to determine the no of bacteria
ii) test to reveal the presence of coliform bacteria
Presumptive, confirmed, completed test
 Major Group of Faecal Pollution Indicators -
There are three major groups.
1. The coliform groups - typified by Escherichia coli.
2. The faecal streptococci group - typified by
Streptococcus faecalis.
3.The sulphite reducing clostridia group - typified by
Clostridium perfringenes
Coliforms

The term coliforms refers to Gram - negative,

non sporing rod shaped bacteria, capable of aerobic
and facultatively anaerobic growth in the presence of
bile salts or other surface active agents with similar
growth - inhibiting properties, which are able to
ferment lactose with the production of acid and gas
within 48 hours at 37C. They are oxidase negative.
The definition includes the genera Escherichia,
Citrobacter; Enterobacter and Klebsiella. Coliform
group is sometimes referred to as total coliforms to
differentiate them from faecal coliforms.
 Faecal Streptococci - Certain streptococcal species of faecal
origin are classified as faecal streptococci. They belong to two
main groups. The first group includes Streptococcus faecalis,
S. faecium and S. durans which are normally present in man
and various animals.
The second group comprises S. bovis. S. equinus and S. avium
which do not usually occur in man. Apart from these species,
other serologically distinct streptococci occasionally present in
faeces are S. mitis and S. salivarius.
Clolstridium Perfringens - Clostridium perfringens is the
most important member of the group, anaerobic sulphite
reducing clostridia. It is normally present in human and animal
faeces, but in much fewer number than E. coli and faecal
streptococci. Thus, it is less sensitive as a direct indicator of
faecal pollution.
The resistant spores of C. perfringens survive in water and in
the environment for much longer time than E. coli and other
faecal indicator organisms.
MO other than coliform bacteria
Besides coliform bacteria fecal streptococci are used as indicators of
fecal contamination
S.faecium, S. bovis are present in large intestine, their occurrence in
water is indicative of fecal pollution.
Slime forming bacteria are capable of eloborationg gummy or
mucilaginous substance either as capsular structures or as extracellular
excretion products.
Iron bacteria transform soluble iron compounds to insoluble iron (ferric
hydroxide) and may be deposited in a sheath around the organism.
Sulfur bacteria oxidise sulphur to sulphuric acid and some bacteria
produce H2S.
Algae produce turbidity, and discoloration.
Viruses : enteroviruses
Detection of Coliform org

Muliple tube fermentaion technique

Presumptive
Confirmed
Completed
Membrane filtration technique
Colilert technique
Coliform-test-technique or Mutiple tube
fermentation test
Coliform-test-technique (or MTFT) is a standard
method followed all over the world to determine
whether the water is potable or faecally polluted.
The technique involves three successive steps,
namely, presumptive test, confirmed test, and
completed test.
In the presumptive test, lactose broth tubes are
inoculated with three different water volumes to give
an estimate of the most probable number, green
lactose bile broth in the confirmed test, and positive
tubes are used to calculate the most probable number
(MPN) value. Presence of coliform bacteria in water
samples are established in completed tests.
Media For Multiple Tube Test

Presumptive test Confirmed test

Group being tested

Lauryl tryptose broth

Mac conkey broth Brilliant green lactose
Total coliforms
bile broth.

Lauryl tryptose broth

Faecal coliforms E.C. broth
Mac conkey broth

Pfizer seletive
Faecal streptococci Azide dextrose broth
enterrococcus agar
Presumptive Test

A series of lactose broth or lauryl sulphate tryptose

broth fermentation tubes are inoculated with measured
amounts of water and incubated at 35C for 24 to 48
hours.

The formation of gas in the inverted vial in the

fermentation tube within 48 hours indicates a positive
presumptive test. Absence of gas formation at the end
of 48 hours constitutes a negative presumptive test.
This means that the water sample does not contain
coliforms and is considered safe.
Confirmed test
Sometimes a false positive presumptive test is obtained. This may be due
to the presence of yeasts, certain Clostridium species and some other
organisms, or by bacterial associations or synergism.

In order to be certain that gas-production is due to coliforms a confirmed

test must be performed.
Two procedures are normally employed. In one method a drop of culture
from a positive lactose broth tube is transferred to brilliant green lactose
bile fermentation broth, and is incubated for 24 to 48 hours at 35C.

The appearance of gas within 48 hrs constitutes a positive confirmed test.

The dye inhibits gram positive organisms and eliminates a false
presumptive test and the syngergistic reaction of gram positive and gram
negative organisms growing together.
In the second method a drop of culture from the positive lactose broth is
streaked on a petriplate containing, Endo's, or eosin-methylene blue agar.
The appearance of nucleated colonies, with or without a metallic sheen,
within 24 hours indicates a positive confirmed test.
Completed test

Isolated colonies from petriplates are transferred into

lactose fermentation broth and streaked on to an agar
slant. The presence of gas in the fermentation broth
and the presence of gram negative non spore forming
bacilli on the slant give evidence that coliform
bacteria were present in the original water sample.
Membrane-Filter-Technique
This technique used for bacteriological examination of water to
determine its potability is being considered advantageous over
coliform-test technique because of its significant advantages.
In this technique, a thin membrane filter-disc is used.
The filter-disc consists of cellulose derivatives and can retain
on its surface all bacteria from the water sample.
The water is filtered through filter-disc and the disc is then
transferred with a sterile forcep on to a thin absorbent pad that
has previously been saturated with the appropriate medium
(generally Endo-broth (GM-9) medium) and accommodated
within a Petri dish.
The Petri dish containing absorbent pad and filter-disc is
incubated at 37C for 18-24 hours.
The medium diffuses through the pores of the filter-disc and
provides nutrient to the bacteria. After the incubation is over,
one can see colonies developing upon the filter-disc. The
characteristic colonies of different bacteria could now be
studied to determine water potability.
 Advantages
1. A large volume of water can be analyzed in a short period of
time without much expenses.
2. The membrane filter-disc can be transferred from one
medium to another to differentiate organisms.
3. Quantitative estimations of certain bacterial types, e.g.,
coliforms can be done using appropriate selective media even
when the bacterial types in question are present in small
numbers.
4. This technique requires much less equipment and, therefore,
can be operated direct in the field.

Disadvantages
1. High turbidity waters limit volume sampled.
2. High population of background bacteria result in overgrowth
3. Metals and phenolic compounds can absorb to filters and
inhibit growth.
Most probable number

In this procedure, water in 10 ml, 1 ml and 0.1 ml

amounts is inoculated into lactose broth tubes. The
tubes are incubated and coliform organisms may be
identified by their production of gas from the lactose.

By referring to a MPN table, a statistical range of the

number of coliform may be determined by observing
how many broth tubes showed gas. This method does
not detect total number of bacteria in the water nor it
locate noncoliforms like Salmonella. However it
indicates the presence and quantity of coliforms.
Standard Plate Count

Water sample is diluted in sterile buffer. Measured
amounts are pipetted into petri dishes. Agar medium is
added and plates incubated. Colony counts are made
and multiplied by the reciprocal dilution factor to have
total bacteria per ml water.
 Advantages of MPN Techniques
1. Interpretation of the results requires minimal experience and
training as results can be got by simply observing for the
presence of gas or no gas.
2. Water samples with high turbidity can be analyzed, since
there is no apparent deleterious effect.
3.Because of the dilutions used in the range of 1:0 or 1:100,
toxic substances present in the sample can be diluted out.
4. MPN technique is the effective method for analyzing
samples such as muds, sludges, sediments etc.
Disadvantages of MPN Techniques
1. MPN procedure takes very long time for the confirmed test
result.
2. In MPN the results are probability calculations and can
not be accurate.
3. MPN requires more glass wares and media.
4. False positive results are of common occurrence.

Membrane Filter
Comparatively smaller quantities of
media required.
Membrane filter is expensive.
More accurate procedure especially at
low levels.
Not suitable for turbid waters as they may
clog the membrane.
More convenient procedure and less
manipulation is required.
Incubation requires 24 hours.
Suitable for field tests
MPN technique
Larger quantities of culture media and
glasswares are required. Sterilization
may need a larger autoclave.
Uses comparatively less expensive
materials.
Statistically based method and there is a
possibility of errors especially at low
levels.
Useful in analyzing turbid water samples.
Less convenient as large quantities of
mateirals are required.
Incubation requires 48-72 hours.
Not suitable for field tests.
Colilert technique

Defined substrate technology describes the technology

whereby the presence of microorganisms possessing a specific
enzyme(s) may be detected. Each enzyme is able to metabolise
a specific substrate. For example, the enzyme b-D-
galactosidase metabolizes b-galactopyranoside and the enzyme
b-glucuronidase metabolizes b-D-glucuronide.
When specific substrates are attached to a chromogen and are
included in a growth medium, microorganisms possessing the
target enzyme (where present) will metabolise the substrate,
resulting in release of the chromogen. The release of
chromogen manifests itself in a colour change of the substrate.
Microorganisms other than the target cannot grow and
metabolise and do not affect the test. A major characteristic of
this technology is that more than one enzyme can be assayed at
the same time providing that each is attached to a chromogen
of a different colour.
 On blood agar plates, C. perfringens grown
anaerobically produces -haemolytic, flat, spreading,
rough, translucent colonies with irregular margins. A
Nagler agar plate, containing 5-10% egg yolk, is used
to identify strains that produce -toxin, a diffusible
lecithinase that interacts with the lipids in egg yolk to
produce a characteristic precipitate around the
colonies.
IMViC reactions

E. coli and A. aerogenes are normally referred to as

faecal and non-faecal contaminants of water,
respectively, and are the most important organisms of
the coliform group. Since they closely resemble each
other in their morphological and cultural characteristics,
biochemical tests, are, performed to differentiate them.
The name IMViC reactions was coined by Parr taking
the first letters of each of the four biochemical tests (I =
indole, M = methyl red, Vi = Voges-Proskauer reaction,
and C = citrate).
 Indole test
In this test, the organism under consideration is grown in peptone Water
broth. It contains tryptophan, which under the action of enzyme
tryptophanase is converted to an Indole molecule, pyruvate and carbon
dioxide. The indole is then extracted from the broth by means of xylene.
To test the broth for indole production, Kovac's reagent is added. A
positive result is indicated by a pink/red layer forming on top of the liquid.
Methyl Red test
VogesProskauer test
These tests both use the same broth for bacterial growth. The broth is
called MRVP broth. After growth, the broth is separated into two different
tubes, one for the Methyl Red (MR) test and one for the Voges-Proskauer
(VP) test. The pH indicator Methyl Red is added to one tube and a red
color appears at pH's lower than 4.2, and indicated positive test. The VP
test uses alpha-naphthol and potassium hydroxide to indicate a positive or
negative test.
Citrate Test
This test uses Simmon's citrate agar to determine the ability of a
microorganism to use citrate as its sole carbon source. The citrate agar is
green before inoculation, and turns blue as a positive test indicator.
IMViC reactions

Test E. coli Enterobacter

I. Indole (ability to Does Does not

produce indole)
II. Methyl red (amount of Produces a pH below 4.5 Does not produce such
acidity produced in a which turns indicator pH and hence indicator
special glucose-broth red. does not turn red. It
medium and detected remains yellow.
by the indicator methyl-
red)
III. Voges-Proskauer Acetylmethylcarbinol not Acetylmethylcarbinol
reaction (ability to produced produced
produce
acetylmethylcarbinol in
a glucose peptone
medium)
IV. Citrate (utilization of Does not grow in a Grow in a medium in
sodium citrate as medium in which which sodium citrate is
source of carbon) sodium citrate is the the only carbon source
only carbon source
Water borne disease

Any disease that can spread through contaminated

water. The contamination can involve bacterial, viral or
protozoan organisms. Some examples of
waterborne diseases include cholera (bacteria),
dysentery (bacteria or amoeba), cryptosporidiosis
(protozoa), hepatitis A (virus) and giardia (protozoa).
Infection can result not only from drinking the water but
also from swimming in the water where it can enter the
body in other ways such as through broken skin.
 Diseases Transmitted by Water
Water is infact a vehicle for the transfer of a wide
range of diseases of microbial origin. The important
bacterial diseases include typhoid fever, cholera and
bacterial dysentry that are generally transmitted when
human feces from carriers or patients contaminates the
water.

Viral diseases transmitted by water include virus A

hepatitis and polio. Many protozoa form cysts which
survive for long periods in water. The causal agents of
amoebiasis, giardiasis etc. are important concerns in
water pollution.
Types

Waterborne pathogens include viruses (e.g., hepatitis

A, poliomyelitis); bacteria (e.g., cholera, typhoid,
coliform organisms); protozoa (e.g.,
cryptosporidiosum, amebae, giardia); worms (e.g.,
schistosomia, guinea worm); and toxins (e.g., arsenic,
cadmium, numerous organic chemicals).
Typhoid fever Salmonella typhi

Paratyphoid fever S. Paratyphosa, S.

shottmuelleri, S. typhimurium

Cholera Vibrio comma

Bacillary dysentery Shigella sp.

Amoebic Dysentery Entamoeba histolytica

Poliomyelitis Polio virus

Viral (infectious) hepatitis Hepatitis viruses

Vasilyev-Weil disease or Leptospira interrogans

infectious jaundice
Protozoal infection
Disease and
Microbial Agent
Transmission
Protozoan (Entamoeba
Amoebiasis
histolytica) (Cyst-like
(hand-to-mouth)
appearance)
Cryptosporidiosi Protozoan
s (oral) (Cryptosporidium parvum)
Protozoan parasite
Cyclosporiasis
(Cyclospora cayetanensis)
Giardiasis (oral- Protozoan (Giardia lamblia)
fecal) (hand-to- Most common intestinal
mouth) parasite
Protozoan phylum
Microsporidiosis (Microsporidia), but closely
related to fungi
Sources of Agent in Water
General Symptoms
Supply
Abdominal discomfort, fatigue,
Sewage, non-treated drinking
weight loss, diarrhea, bloating,
water, flies in water supply
fever
Flu-like symptoms, watery
Collects on water filters and
diarrhea, loss of appetite,
membranes that cannot be
substantial loss of weight,
disinfected, animal manure,
bloating, increased gas,
seasonal runoff of water.
nausea
cramps, nausea, vomiting,
Sewage, non-treated drinking
muscle aches, fever, and
water
fatigue
Untreated water, poor
disinfection, pipe breaks,
leaks, groundwater
contamination, campgrounds Diarrhea, abdominal
where humans and wildlife discomfort, bloating, and
use same source of water. flatulence
Beavers and muskrats create
ponds that act as reservoirs
for Giardia.
The genera of
Encephalitozoon intestinalis Diarrhea and wasting in
has been detected in immunocompromised
groundwater, the origin of individuals..
drinking water [3]
Parasitic infections
Disease and Microbial Sources of Agent in Water
General Symptoms
Transmission Agent Supply
Members of Fresh water contaminated Rash or itchy skin. Fever,
Schistosomiasis
the genus with certain types of snails chills, cough, and muscle
(immersion)
Schistosoma that carry schistosomes aches
Dracunculiasis Allergic reaction, urticaria
Dracunculus Stagnant water containing
(Guinea Worm rash, nausea, vomiting,
medinensis larvae
Disease) diarrhea, asthmatic attack.
Intestinal disturbances,
Tapeworms of
Drinking water contaminated neurologic manifestations,
Taeniasis the genus
with eggs loss of weight,
Taenia
cysticercosis
GIT disturbance, diarrhea,
Drinking water contaminated
Fasciolopsis liver enlargement,
Fasciolopsiasis with encysted
buski cholangitis, cholecystitis,
metacercaria
obstructive jaundice.
Hymenolepiasis
Abdominal pain, anorexia,
(Dwarf Hymenolepis Drinking water contaminated
itching around the anus,
Tapeworm nana with eggs
nervous manifestation
Infection)
 Liver enlargement, hydatid
Drinking water contaminated cysts press on bile duct and
Echinococcosis Echinococcus
with feces (usually canid) blood vessels; if cysts rupture
(Hydatid disease) granulosus
containing eggs they can cause anaphylactic
shock
multiceps contaminated drinking water
coenurosis increases intacranial tension
multiceps with eggs
Mostly, disease is
asymptomatic or accompanied
Drinking water contaminated by inflammation, fever, and
Ascaris
Ascariasis with feces (usually canid) diarrhea. Severe cases involve
lumbricoides
containing eggs Lffler's syndrome in lungs,
nausea, vomiting, malnutrition,
and underdevelopment.
Peri-anal itch, nervous
Enterobius Drinking water contaminated
Enterobiasis irritability, hyperactivity and
vermicularis with eggs
insomnia

Morbidity Mortality
Disease
(cases per year) (deaths per year)
1,500,000,000 100,000
Schistosomiasis 200,000,000 200,000
Bacterial infection
Disease and
Sources of Agent in Water
Transmis Microbial Agent General Symptoms
Supply
sion
Dry mouth, blurred and/or
Bacteria can enter a wound from double vision, difficulty
contaminated water sources. swallowing, muscle
Can enter the gastrointestinal weakness, difficulty
Botulism Clostridium botulinum
tract by consuming breathing, slurred speech,
contaminated drinking water vomiting and sometimes
or (more commonly) food diarrhea. Death is usually
caused by respiratory failure.
Most commonly Produces dysentery like
Campylobac caused by Drinking water contaminated symptoms along with a high
teriosis Campylobacter with feces fever. Usually lasts 210
jejuni days.
In severe forms it is known to be
one of the most rapidly fatal
illnesses known. Symptoms
include very watery
Spread by the
Drinking water contaminated diarrhoea, nausea, cramps,
Cholera bacterium Vibrio
with the bacterium nosebleed, rapid pulse,
cholerae
vomiting, and hypovolemic
shock (in severe cases), at
which point death can occur
in 1218 hours.
 Mostly diarrhea. Can
cause death in
immunocompromised
Certain strains of
Water contaminated individuals, the very
E.coli Infection Escherichia coli
with the bacteria young, and the
(commonly E. coli)
elderly due to
dehydration from
prolonged illness.
Naturally occurs in Symptoms include
water, most cases lesions typically
from exposure in located on the
swimming pools or elbows, knees, and
M. marinum Mycobacterium more frequently feet (from swimming
infection marinum aquariums; rare pools) or lesions on
infection since it the hands
mostly infects (aquariums). Lesions
immunocompromised may be painless or
individuals painful.
Caused by a number of
Frequent passage of
species in the genera
feces with blood
Shigella and Water contaminated
Dysentery and/or mucus and in
Salmonella with the with the bacterium
some cases vomiting
most common being
of bloo
Shigella dysenteriae
 Pontiac fever produces
milder symptoms
resembling acute
influenza without
pneumonia.
Caused by bacteria
Legionnaires disease
Legionellosis (two distinct belonging to genus Contaminated water: the
has severe symptoms
forms: Legionnaires Legionella (90% of organism thrives in
such as fever, chills,
disease and Pontiac cases caused by warm aquatic
pneumonia (with
fever) Legionella environments.
cough that sometimes
pneumophila)
produces sputum),
ataxia, anorexia,
muscle aches, malaise
and occasionally
diarrhea and vomiting
Begins with flu-like
symptoms then
resolves. The second
Water contaminated by
Caused by bacterium of phase then occurs
Leptospirosis the animal urine
genus Leptospira involving meningitis,
carrying the bacteria
liver damage (causes
jaundice), and renal
failure
Caused by a number of Swimming in water Ear canal swells causing
Otitis Externa (swimmers
bacterial and fungal contaminated by the pain and tenderness to
ear)
species. responsible pathogens the touch
 Drinking water
Samanellos Caused by many bacteria contaminated with the
is of genus Salmonella bacteria. More common
as a food borne illness.
Ingestion of water
Typhoid
Salmonella typhi contaminated with feces
fever
of an infected person
Can enter wounds from
contaminated water.
Vibrio vulnificus, Vibrio
Vibrio Also got by drinking
alginolyticus, and Vibrio
Illness contaminated water or
parahaemolyticus
eating undercooked
oysters.
Symptoms include
diarrhea, fever, vomiting,
and abdominal cramps
Characterized by sustained
fever up to 40C
(104F), profuse
sweating, diarrhea, less
commonly a rash may
occur. Symptoms
progress to delirium and
the spleen and liver
enlarge if untreated. In
this case it can last up to
four weeks and cause
death.
Symptoms include
explosive, watery
diarrhea, nausea,
vomiting, abdominal
cramps, and
occasionally fever.
Viral infection
Sources of
Disease and Agent in
Microbial Agent General Symptoms
Transmission Water
Supply
Symptoms include
Manifests itself
Adenovirus common cold
Adenovirus in improperly
infection symptoms, pneumonia,
treated water
croup, and bronchitis
Symptoms include
Astrovirus, Calicivirus, Manifests itself diarrhea, nausea,
Gastroenteritis Enteric Adenovirus, in improperly vomiting, fever,
and Parvovirus treated water malaise, and
abdominal pain
Symptoms include fever,
SARS (Severe
Manifests itself myalgia, lethargy,
Acute
Coronavirus in improperly gastrointestinal
Respiratory
treated water symptoms, cough, and
Syndrome)
sore throat
 Symptoms are only acute (no
chronic stage to the virus) and
Hepatitis Hepatitis A virus Can manifest itself in water (and include Fatigue, fever,
A (HAV) food) abdominal pain, nausea,
diarrhea, weight loss, itching,
jaundice and depression.
90-95% of patients show no
symptoms, 4-8% have minor
symptoms (comparatively)
with delirium, headache, fever,
Poliomyel
Enters water through the feces of and occasional seizures, and
itis Poliovirus
infected individuals spastic paralysis, 1% have
(Polio)
symptoms of non-paralytic
aseptic meningitis. The rest
have serious symptoms
resulting in paralysis or death
BK virus produces a mild
respiratory infection and can
infect the kidneys of
Polyomav Very widespread, can manifest immunosuppressed transplant
Two of
irus itself in water, ~80% of the patients. JC virus infects the
Polyomavirus: JC
infectio population has antibodies to respiratory system, kidneys or
virus and BK virus
n Polyomavirus can cause progressive
multifocal
leukoencephalopathy in the
Water purification

To be potable water must be more or less free from

turbidity, colour, taste and odour well aerated and
most important of all it must be free from pathogenic
microorganisms and harmful chemicals.
Wells and springs etc. are the sources of most of the
water supply for individual families in rural areas. As
the water of wells and springs penetrates through the
layers of soil it undergoes filtration which removes
suspended particles including microorganisms.
The precaution of prime importance in this regard is
to ensure the supply of non-polluted water is that such
water resources should strictly be located at a safe
distance from possible sources of contamination
Municipal water purification
Four main processes are employed in a municipal water purification system to
ensure the supply of non-polluted drinking water. These processes are :
flocculation, sedimentation, filtration and disinfection.
 Flocculation
Water after pumping from raw water reservoir (natural
sources) is collected in large tanks/basins for a sufficient time
period to permit large particulate matter to settle down at the
bottom.
This material is removed and then the water is treated with
flocculants such aluminium sulphate, allum, sodium alluminate
colloidal silicate, calcium or Bentonite which form a floc that
precipitates and carriers with it microorganisms on the surface
suspended organic matter settle onto the bottom of the
tanks/basins.
Sedimentation
The water, after coagulation, is left in settling basin further for
sufficient period to allow sedimentation of remaining
materials.
Sedimentation however considerably reduces microbial
population of the water aside from removing most of the
suspended particles.
 Filtration
After sedimentation, water is subjected to sandfilters to remove
flocks of living organisms. The process of filtration is highly
critical and important as it can remove protozoan cysts and
also about 98-99% of bacteria from water.
The water may also be filtered through activated charcoal to
remove potentially toxic organic compounds and organic
compounds that impart undesirable colour and/or taster to the
water.
Slow Sand Filters These sand Filters comprise of layers of
fine sand, fine and coarse gravel. The floor at the base is made
of tiles provided with perforations for intake of filtered water.
The uppermost fine sand layer acts as a biological filter as its
interspaces are clogged by colloidal floccules in water,
encapsulated microorganisms, and algae, and retains bacteria
making the water free from them. The efficiency of filter is
further enhanced as the negatively charged bacteria are held by
the positively charged colloidal material in the layer. This filter
normally removes 98% of the bacterial population present in
water.
Rapid Sand Filters This method involves essentially the same
equipments as the slow sand filters but the sand filter area is
much smaller with provision for frequent back washing.
Disinfection
Disinfection is the final step is municipal water purification
and it ensures that no pathogenic microorganisms are carried
through water. For water supplies of small towns and localities
sodium or calcium hypochlorite may be used to disinfect
water, but for larger cities, however, chlorination (use of
chlorine) has been the traditional method for disinfection.
Chlorination often removes bacterial contamination since
chlorine reacts with water to yield hypochlorous and
hypochloric acids which are potent microbicides. Chlorine is
obtained as liquid under pressure but releases into water as gas.
It easily dissolves in water and, in addition to killing the
microbes, it is also effective in oxidizing organic matter.
Microbiology of sewage

Sewage is the used water supply containing domestic

waste together with human excrement and wash water
and industrial waste, including acids, greases, oils,
animal matter, vegetable matter and storm waters.

The basic principle in sewage treatment is that water is

separated from the waste while the solid organic
matter is biodegraded by microorganisms to simple
compounds like nitrates, sulphates, carbonates, carbon
dioxide, methane etc.
Sewage treatment is managed on small scale as in
individual homes and rural areas as well as an large
scale in towns by municipal bodies.
Sewage composition
a) Chemical Composition
The sewage consists of approximately 99% water and 1% inorganic and
organic matter in suspended and soluble forms.
Lignocellulose, cellulose, proteins, fats, and various inorganic particulate
matter exist in suspended state, whereas sugars, fatty acids, alcohols,
amino acids, and inorganic ions constitute the soluble forms.
The sewage of towns in our country contains about 350 ppm biodegradable
organic matter, 52 ppm N2, 45 ppm potassium and 16 ppm phosphorus.
Salts of several heavy metals such as Zn, Cr, Ni, Pb, etc. are also present
above permissible levels in sewage.
(b) Microbial Composition
The microbial population per millilitre of sewage may vary from a few lacs
to several millions.
Various types of microorganisms, viz., microfungi, bacteria and protozoa,
collectively called sewage fungus , are known to grow profusely in
sewage.
In addition, viruses and many microalgal genera have also been recorded
from sewage. Bacteria occurring in sewage are mainly intestinal and soil
inhabiting and their common types are coliforms, streptococci, clostridia,
micrococci, Proteus, Pseudomonas, and lactobacilli.
Sewage classification

Sewage may be classified mainly into two types,

namely, domestic and industrial.
All household wastes and human and animal excrete
constitute domestic sewage, whereas the industrial
wastes constitute industrial sewage.
Since industrial wastes vary greatly in their
composition (some may be highly alkaline such as
soda wastes, some highly acidic such as acid-mine
drainage, and others toxic because of presence of
heavy metals, antibiotics, pesticides, etc.), the
treatment of industrial sewage proves highly difficult
in comparison to domestic sewage.
Characteristic of sewage
(a) Biochemical Oxygen Demand (BOD) and Oxygen
Consumption (OC) values are extremely high in sewage.
(b) The sewage organic matter undergoes anaerobic or partial
decomposition resulting in the production of obnoxious gases,
namely CH3, CO and H2S due to anoxic condition. Besides
being toxic, these gases react with water and produce acids.
(c) Production of acids in large quantity make the sewage more
acidic thus making it unfit for supporting life activities.
(d) Heavy metals are generally present in abnormal
concentration in sewage.
All these characteristics of sewage, viz., condition, high
acidity, high heavy metal concentration, and reduced
photosynthetic rate due to poor illumination cause death of
oxygen-dependent organisms such as aerobic microorganisms,
plants and animals in sewage. This is the reason why sewage is
dominated by organisms capable of growing in anaerobic
environments.
Sewage disposal
Sewage disposal has become of prime importance
now-a-days as it brings undesirable and harmful
effects on living beings.
Untreated or inadequately treated sewage is generally
disposed of into natural water reservoirs
It is necessary, therefore, that the sewage must be
treated before its disposal so that we can, on one hand,
save organisms including men from bad effects and,
on the other hand, can utilized it to the maximum for
our welfare.
Disposal of sewage as such or inadequately treated
one, generally leads to following consequences:
Frequent dissemination of water-borne disease causing
microorganisms in large number
 BOD : Biochemical Oxygen Demand (BOD; sometimes called Biological
Oxygen Demand) represents the amount of dissolved oxygen needed by
the microorganisms in a particular amount of water at 20C in 5 days.

The strength of sewage is expressed in terms of BOD. The magnitude of
BOD is related to the amount of organic material present in the sewage. A
medium or strong sewage (having more oxidisable matter) has high BOD
value, and a weak sewage (having less oxidisable matter) has low BOD
value. Thus, BOD determinations provide the best information concerning
the strength of sewage and the efficiency of sewage treatment processes.
Oxygen Consumption
OC represents the amounts of dissolved oxygen utilized by biotic
communities for the oxidative decomposition of organic matter in one liter
of water in one hour. Dissolved oxygen in sewage becomes totally depleted
because of high oxygen demand and low photosynthetic rate.
Photosynthesis in sewage comes down because of poor illumination as the
suspended solids obstruct sun light. However, the depletion of dissolved
oxygen leads to anoxic (oxygenless) condition.
Sewage trt

The objectives behind the sewage treatment would be to kill

pathogenic microorganisms prevent anoxia, raise the pH to
alkaline side, increase photosynthetic rate, reduce organic
content, etc.
Single Dwelling Unit Treatment Processes
Outdoor Toilets
Septic Tanks
These are used for residential quarters. All the residential
sewage is passed through suitable pipes leading to a tank
located to a tank at a suitable place and made of metal or
concrete. The septic tank device should be so fitted that the
sewage does not drain by any chance into water supply of the
residence.
 Municipal Treatment Processes

Municipal sewage treatment systems carry out various steps
involved. These steps are, namely primary (or mechanical)
treatment, secondary (or biological) treatment, and tertiary (or
final) treatment.
A. Primary (or Mechanical) Treatment
When the sewage arrives at a sewage treatment plant, it is first
subjected to mechanical (or physical) means. viz., flowing
dilution and sedimentation to remove its coarse solid materials.
The sewage is passed through a series of filters of graded
opening and then allowed to flow through sedimentation units
(tanks, basins, etc.). Coarse solid materials are concentrated in
and collected from sedimentation units; these particulate
materials are collectively called sludge. Following
sedimentation, the sludge and liquid affluent are processed
separately during secondary treatment.
 Secondary (or Biological) treatment
This is purely a biological treatment of mechanically treated sewage
and concerns microbial activity which biodegrades organic
substrates and oxidisable inorganic compounds.
This treatment accomplishes two important phases, namely, aerobic
phase and anaerobic phase. The aerobic phase consists of aerobic
digestion of sludge by various filters (e.g., trickling filters),
oxidation ponds and activated sludge process, and the anaerobic
phase is represented by anaerobic digestion of sludge.
Anaerobic Phase of Secondary Treatment
Anaerobic Digestion of Sludge. The sludge collected after primary
(mechanical) treatment of sewage is subjected to anaerobic (oxygen-
free) digestion is separate tank. Since anaerobic condition prevails in
this tank, the anaerobic microbes bring about digestion of organic
matter by degrading them to soluble substances and gaseous
products (methane, 60-70%; CO2, 20-30%; and smaller amounts of
H2 and N2). This gas mixture can be used for operating power for
the sewage plant or as a fuel.
 Tertiary (or Final) Treatment
Since the sewage-effluent treated during secondary treatment
process still contains non biodegradable organic pollutants (if
sewage contains industrial wastes) and mineral nutrients
particularly nitrogen and phosphorus salts, it is subjected to
tertiary (or final) treatment for their removal.
The sewage effluents containing nitrogen and phosphorus salts
can cause serious eutrophication in aquatic ecosystems.
Nonbiodegradable organic pollutants are normally removed
by using activated carbon filters whereas phosphorus and
nitrogen salts by chemical treated.
The find step of tertiary treatment is disinfection which is
commonly accomplished by chlorination using either sodium
or calcium hypochlorite or chlorine. Now the effluent is a
clean water and is considered microbiologically safe even for
human consumption
 Standards for Bacteriological Quality of Public Drinking Water
Supplies
If the required continuous disinfection is not being provided, an
immediate boil water advisory is recommended.
Bacteriological samples shall be taken at representative locations in
the distribution system and must include the beginning and the end
of the system, in accordance with the recommended number of
samples.
Disinfection residuals must be checked and recorded for each
sampling point. For potable water dispensing units, disinfection
residuals need not be checked.
If no disinfectant residual is detected at the sampling location, the
operator of the system shall be advised of the fact that no residual is
detected, and in consultation with the appropriate authorities and in
accordance with existing guidelines, remedial action shall be
initiated. This may include the issuing of a boil water advisory for
the area affected by the lack of disinfectant residual.
Microbiological Water Quality Standard
Guidelines for bacteriological water quality differs from
country to country, generally keeping with those recommended
by World Health Organization. Usually coliform counts are
used to setup water quality standards.
It should be noted that standards set using coliform counts are
arbitrary and they represent a measure of degree of possible
human faecal contamination. The number of coliforms
permitted in water is dependent on the purpose for which the
water is used. The standards for drinking water are more
stringent than those for recreational waters

1.E.coli should not be detectable in any sample of 100ml

2. no sample of 100ml should contain more than three coliform
organisms.
3.coliform organisms should not be detectable in any two
consecutive samples of 100ml from the same or a closely
related sampling point.
4.for any given distribution system, coliform organisms should
not occur in more than five percent of routine samples,
provided that atleast 50 samples have been examined at regular
intervals throughout the year.
BACTERIOLOGICAL STANDARDS FOR DRINKING WATER

Parameter Standard
Pathogenic Organisms
(includes E. coli)
Water must be free from pathogenic
organisms and organisms indicating fecal
contamination, such as fecal coliform
bacteria, Escherichia coli bacteria,
enterococci bacteria and coliphage
viruses.
Total Coliforms Water must not contain more than
10 total coliforms per 100 millilitres
of water collected.
Total Coliforms When collected, at least 90% of
samples must be free from total
coliform bacteria.
Facultatively Aerobic or
Anaerobic Heterotrophic Bacteria
Water must not contain more than 500
facultatively aerobic or anaerobic
heterotrophic bacteria per millilitre of
water collected.
 If the bacteriological test reveals the presence of E.coli, re-sampling of
the site, as well as up and downstream locations, should be carried out
within 24 hours.
However, a boil water advisory may be recommended before the re-
sampling results are known depending upon the extent of E.coli
contamination in the initial testing, combined with knowledge of other
problems pertaining to the water system.
Regardless, if the repeat test reveals the presence of E.coli, a boil water
advisory is recommended.
If the bacteriological test reveals the presence of total coliforms, but no
E.coli, re-sampling of the site, as well as up and downstream locations,
should be carried out as soon as is practically possible.
If the consecutive test confirms the presence of total coliforms, but no
E.coli, a boil water advisory is recommended for water systems that
have only disinfection but no additional water treatment (such as
coagulation, sedimentation, filtration or equivalent technologies) or
have no significant operational procedures and controls over the water
system. Regardless, remedial action shall commence in consultation
with the water system operator.