BACTERIOLOGY OF WATER

AND ANALYSIS

basics
Dr.T.V.Rao MD

Dr.T.V.Rao MD @ Rao's Microbiology

The Quality of Water is

polluted with many
Factors

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Bacteriology of water to
identify the bacterial
contamination
The bacteriological examination of water

is performed routinely by water utilities

and many governmental agencies to
ensure a safe supply of water for
drinking, bathing, swimming and other
domestic and industrial uses. The
examination is intended to identify water
sources which have been contaminated
with potential disease-causing
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How the water gets contaminated

Such contamination generally occurs
either directly by human or animal feces,
or indirectly through improperly treated
sewage or improperly functioning sewage
treatment systems. The organisms of
prime concern are the intestinal
pathogens, particularly those that cause
typhoid fever and bacillary dysentery
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Common Diseases Spread by Water

Typhoid fever
Cholera
Diarrhoeal
diseases
Polio myelitis
Viral hepatitis A
and E

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Contamination Happens
When water comes in
Contact

Faecal
matter
Sewage
contaminati
on

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How the Water Contaminated

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Natural Water contaminated with

Pseudomonas spp
Flavobacterium
Chromobacterium
Acinetobacter spp

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Water we drink should be Free from

Should be free from pathogenic
bacteria
The primary criteria is the water
should be free from Coliforms if
they are present it indicates
faecal contamination
Thermotolerant E.coli bacteria
indicates the presence of
contamination
Faecal Streptococci thermos
tolerant indicates contamination
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Bacteriological Analysis of Water

Bacteriological
water analysis is a
method of analysing
water to estimate
the numbers of
bacteria present
and, if needed, to
find out what sort of
bacteria they are. It
represents one

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How the bacteriological

analysis of water Helps
It is a microbiological
analytical procedure
which uses samples of
water and from these
samples determines
the concentration of
bacteria. It is then
possible to draw
inferences about the
suitability of the water
for use from these

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What are the Indicator

organisms contaminate water
The common feature of all these routine
screening procedures is that the primary analysis
is for indicator organisms rather than the
pathogens that might cause concern. Indicator
organisms are bacteria such as non-specific
coliforms, Escherichia coli and Pseudomonas
aeruginosa that are very commonly found in the
human or animal gut and which, if detected, may
suggest the presence of sewage
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Methods used in Culturing of

Water
Analysis is usually
performed using culture,
biochemical and
sometimes optical
methods. When indicator
organisms levels exceed
pre-set triggers, specific
analysis for pathogens
may then be undertaken
and these can be quickly
detected (where
suspected) using specific
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culture methods or

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Multiple tube method

One of the oldest methods is called the multiple
tube method. In this method a measured subsample (perhaps 10 ml) is diluted with 100 ml
of sterile growth medium and an aliquot of 10
ml is then decanted into each of ten tubes. The
remaining 10 ml is then diluted again and the
process repeated. At the end of 5 dilutions this
produces 50 tubes covering the dilution range
of 1:10 through to 1:10000.
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Methodology of Bacterial
analysis of Water
The tubes are then incubated at a pre-set temperature
for a specified time and at the end of the process the
number of tubes with growth in is counted for each
dilution. Statistical tables are then used to derive the
concentration of organisms in the original sample. This
method can be enhanced by using indicator medium
which changes colour when acid forming species are
present and by including a tiny inverted tube called a
Durham tube in each sample tube. The Durham inverted
tube catches any gas produced. The production of gas
at 37 degrees Celsius is a strong indication of the
presence of Escherichia
coli.
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Laboratory pictures show

how the Water is Analysed

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Membrane filtration
Most modern laboratories use a refinement
of total plate count in which serial dilutions
of the sample are vacuum filtered through
purpose made membrane filters and these
filters are themselves laid on nutrient
medium within sealed plates. The
methodology is otherwise similar to
conventional total plate counts. Membranes
have a printed millimetre grid printed on
and can be reliably used to count the
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Pour plate method

When the analysis is looking for bacterial
species that grow poorly in air, the initial
analysis is done by mixing serial dilutions of the
sample in liquid nutrient agar which is then
poured into bottles which are then sealed and
laid on their sides to produce a sloping agar
surface. Colonies that develop in the body of
the medium can be counted by eye after
incubation.
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Pour plate method

The total number of colonies is referred
to as the Total Viable Count (TVC). The
unit of measurement is cfu/ml (or colony
forming units per millilitre) and relates to
the original sample. Calculation of this is
a multiple of the counted number of
colonies multiplied by the dilution used.
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PLATE COUNT
Test of water contamination in which the number of
the colonies of coliform-bacteria Escherichia coli (E.
coli) per 100 milliliter of water is counted. The result
is expressed as 'Coliform Microbial Density' and
indicates the extent of fecal matter present in it.
According to common water quality standards
drinking water must be completely free from any
colony, bathing and swimming pool water can have
about 200 colonies, and recreational (fishing and
boating) water about 1000 colonies.
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Multiple tube Methods

Prior to the membrane
filter method, the multiple
tube method was used for
bacteriological examination
of water. In conjunction
with this test, a total plate
count was also conducted.
These tests are discussed
here since they are still
considered standard tests
in the water works field.
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Reporting the Results of

Water Analysis
When a water
sample arrives at
the laboratory,
two tests, the
plate count and
the coliform test
by the multiple
tube method, are
made and

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Reporting the Results of Water

Analysis
The coliform test actually
consists of two steps known as
the presumptive test and the
confirmed test. Under certain
conditions, it is necessary to
go one step further and make
a completed test; however,
this step is not always
necessary. To make the tests,
small portions of the water
sample are used in accordance
with the following procedures.
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The Plate Count

The plate count is a test made
by the laboratory to determine
the total number of bacteria
present in the sample. This test
does not differentiate between
the many different types of
bacteria and is thought of as
giving index to the general
"housekeeping" practices. A
"high" count indicates that
some type of contamination is
present and is undesirable.
The test is made by placing a
portion of agar in a petri dish.
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Making the Microbes to

grow on the Petri dish
A portion of the water sample
is placed in the petri dish along
with the agar. It is then placed
in an incubator with the
temperature at 37C or 98.6F,
which is body temperature.
After 24 hours, the plate is
removed, examined, and the
colonies in and on the agar are
counted and recorded on the
report form as "Bacteria per ml
at 37C".
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Types of Nutrient media

used in analysis
MacConkey agar is culture medium
designed to grow Gram-negative
bacteria and stain them for lactose
fermentation. It contains bile salts
(to inhibit most Gram-positive
bacteria), crystal violet dye (which
also inhibits certain Gram-positive
bacteria), neutral red dye (which
stains microbes fermenting
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Presence of Bacteria Means

The presence of the Coliaerogenes group of
bacteria in the above
tests does not definitely
mean that harmful
bacteria are present.
Coliform bacteria are
normally present in great
numbers in the human
intestine and, except in
unusual circumstances,
are not harmful to Dr.T.V.Rao MD @ Rao's Microbiology
humans.

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What the Indication of

presence of Bacteria
When present in a water
sample, they do, however,
indicate the presence of faecal
contamination and the
possibility that harmful
(pathogenic) organisms, such
as typhoid fever germs, may be
present. Therefore, the tests
are not measures of actual
disease-producing organisms,
but rather are indicators of the
possibility that they are
present.
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Public Health Concept

The coliform group
has been used
extensively as an
indicator of water
quality and has
historically led to
the public health
protection concept.
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Popular Methods to
analyse the Water

Routine and widely accepted techniques are

discussed, as are methods which have emerged
from recent research developments. Approved
traditional methods for coliform detection
include the multiple-tube fermentation (MTF)
technique and the membrane filter (MF)
technique using different specific media and
incubation conditions. These methods have
limitations, however, such as duration of
incubation, antagonistic organism interference,
lack of specificity and poor detection of slowDr.T.V.Rao MD @ Rao's Microbiology

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Methods in use for

Bacteriological analysis
The simple and inexpensive membrane filter technique
is the most widely used method for routine enumeration
of coliforms in drinking water.The detection of coliforms
based on specific enzymatic activity has improved the
sensitivity of these methods. The enzymes beta-D
galactosidase and beta-D glucuronidase are widely used
for the detection and enumeration of total coliforms and
Escherichia coli, respectively. Many chromogenic and
fluorogenic substrates exist for the specific detection of
these enzymatic activities, and various commercial
tests based on these substrates are available
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Bacteriological Identification
with molecular methods
The immunological, polymerase chain reaction (PCR)
and in-situ hybridization (ISH) techniques. In the
immunological approach, various antibodies against
coliform bacteria have been produced, but the
application of this technique often showed low antibody
specificity. PCR can be used to detect coliform bacteria
by means of signal amplification: DNA sequence coding
for the lacZ gene (beta-galactosidase gene) and the
uidA gene (beta-D glucuronidase gene) has been used
to detect total coliforms and E. coli, respectively.
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Is the PCR is precise in

analysis of Water
However, quantification with PCR is still lacking in
precision and necessitates extensive laboratory
work. The FISH technique involves the use of
oligonucleotide probes to detect complementary
sequences inside specific cells. Oligonucleotide
probes designed specifically for regions of the
16S RNA molecules of Enterobacteriaceae can be
used for microbiological quality control of
drinking water samples
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Detection of Virus
Methods are available for isolation of
Enterovirus and other cytopathogenic
viruses from water
But are not part of routine tests unless
epidemics happens
However Viruses are destroyed with
Chlorination of water
The Free residual chlorination is at least 0.5
mg per litre for a contact period of 30
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Protozoa in Water
Endamoeba histolytica
Giardia species
Balantidium coli
However there are no
specific tests
Coliforms are not reliable
as indicators of protozoal
contamination

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This Program is made for basic

understanding on Water bacteriology,
for Undergraduate Medical students
Specific Protocols to be followed form
your Laboratory
Email
doctortvrao@gmail.com
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