Examination of water

BACTERIOLOGY OF WATER
DR. ANKUR KUMAR

Introduction
• Drinking water must be
visually acceptable, clear and without any
disagreeable taste or odour.
Should be free from chemical toxins and pathogenic
microorganisms.
• Many major human diseases, e.g, typhoid, cholera, other
diarrhoeal diseases, poliomyelitis and viral hepatitis A
and E are waterborne.
• These pathogens reach water sources through fecal or
sewage pollution

Bacteriology of Water
 Water is said to be contaminated or polluted ,when
it is contaminated with sewage or other
excreted matter from humans and animals
that contains infective and parasitic agents,
poisonous chemical substances, and
industrial or other wastes.

Bacterial Flora in Water
• Bacterial flora in water can be classified
into three groups.
1. Natural water bacteria:
2. Soil bacteria:
3. Sewage bacteria:

 Bacterial Flora in Water
 Natural water bacteria:
• These are the bacteria that are commonly found in
water, free from
gross pollution
• e.g- Micrococcus, Pseudomonas, Serratia,
Flavobacterium, Acinetobacter.
 Soil bacteria:
• These are the bacteria that are not normal inhabitants of
water but are found after being washed into the water
during rains
• e.g- Aerobic spore forming bacilli and Enterobacter
species.

Sewage bacteria:
• These are the bacteria that are not normal
inhabitants of water but are found in water after
being contaminated with sewage
• e.g, E. coli, Clostridium perfringens.
• These bacteria include those which are the normal
inhabitants of the intestine of humans and animals.
• These also include the bacteria that live mainly on
decomposed organic matter of either plant or animal
origin.

Bacterial flora in water
Source of water Bacteria
Natural water bacteria Micrococcus, Pseudomonas,
Acinetobacter, Serratia, Alcaligenes, and
Flavobacterium
Soil bacteria washed into water Bacillus subtilis, Enterobacter cloacae, and
Enterobacter aerogenes
Sewage bacteria:
Proper sewage bacteria, Clostridium perfringens, Proteus species
Intestinal flora through sewage Escherichia coli, Enterococcus fecalis,
Proteus species, Klebsiella species,
Clostridium perfringens

Factors Determining the Number of Bacteria in Water
 Salinity:
 more is the salinity, lesser is the number of bacteria.
However, some halophilic bacteria survive better in saline
water.
 Acidity:
 Acidity of water has a deleterious effect on most of the
bacteria.
 Temperature:
 Body temperature usually favours survival of the bacteria.
However, in the presence of organic materials, bacteria
tend to multiply even at high temperature.
 Light:
 Sunlight with the wavelength of 300–400nm is highly
bactericidal, provided water is clear and static. The
bactericidal effect is reduced due to the presence of
organic matter and due to movement in water.

Factors Determining the Number of Bacteria in Water
 Storage:
decreases bacterial count in stored water due to
sedimentation.
 Organic matter:
When organic matter is plenty, the microorganisms
tend to multiply and are present in large numbers
whereas when it is less, the organisms are few.
 Type of water:
Surface water is more likely to be contaminated than
the deep water. The later is usually pure.

• Water-Borne Microorganisms
 Supply of drinking water is contaminated with sewage or
other excreted matter from humans and animals;
 it can transmit a wide number of pathogens that includes
bacteria, viruses, and parasites.

Water-borne pathogens
Bacteria Vibrio cholerae; Salmonella Typhi,
Salmonella Paratyphi A, B, and C;
Shigella spp.; Escherichia coli;
Yersinia enterocolitica;
and Campylobacter jejuni
Viruses Hepatitis A virus, Hepatitis E virus,
poliomyelitis virus, rotavirus, and
Norwalk virus
Protozoa Entamoeba histolytica, Giardia
lamblia, Cryptosporidium spp.,
Cyclospora spp., Isospora spp., and
Balantidium coli
Helminthes Ascaris lumbricoides, Trichuris
trichiura, and Enterobius vermicularis

Bacteriological Examination of water
 Supplies of drinking water contaminated with sewage or
other excreted matter from man and animals may cause
diseases (such as typhoid, cholera, campylobacteriosis,
amoebiasis and helminthiasis.) there fore need of
bacteriological examination.
 It is impractical to attempt directly to detect the presence
of all different kinds of water borne pathogens, any of
which may be present intermittently.
 Instead, reliance is placed on testing the supply for
microorganisms which indicate that fecal pollution has
taken place.

 The indicators are usually common intestinal
commensal bacteria which are universally present in,
and excreted in large numbers by man and animals and
are rarely found in other sources.
 Their presence indicates that the
fecal matter has entered the water supply,
fecal bacteria have not been killed or removed by
purification process and
supply is therefore liable to contamination with
dangerous intestinal pathogens.

Indicator organisms
 Microorganisms for use as indicators of faecal
pollution should satisfy several criteria.
 They should be present in faeces in greater numbers than
other pathogen yet be unable to proliferate in water to any
extent.
 Moreover, they should be more resistant than pathogens to
the stresses of the aquatic environment and disinfection
processes.
 Usually a number of indicator organisms are sought.

Indicator organisms
1. Coliforms (presumptive
coliforms)
2. Faecal (Thermotolerant)
coliforms
3. E. coli or ‘fecal E. coli’
4. Faecal streptococci
5. Sulphite – reducing clostridia
6. Pseudomonas aeruginosa
7. Bacteriophage that infect
Escherichia coli.

Coliforms (presumptive coliforms)
• are the bacteria that occur in large no. in feces &
sewage, but
• are also found in the environment in the absence
of fecal contamination.
• their presence in the water does not necessarily
signify fecal contamination.
• are the members of family Enterobacteriaceae,
• grow in the presence of bile salts and produce
acid and gas from lactose within 48 hr at 37 0C.

Cont...
• In order to include bacteria that do not ferment
lactose, the definition now applying in the UK
has been amended.
 Now, the coliforms are defined as members of
the Enterbacteriaceae capable of growing at 37
0C that normally possess β- galactosidase.

Cont.....
This change in definition permits –
 Introduction of rapid methods of enzyme
detection to indicate the presence of
coliforms.
 However, the inclusion of ‘normally’ will
cause some β- galactosidase negative strains
to be classified as coliforms.
 As a consequence, more water samples
will now fail because of higher total coliform
counts.

Cont....
The total coliform count is widely regarded as
the most reliable indicator of potable water
quality.

Fecal ( or thermotolerant)
coliforms
Confirm the criteria for coliforms organisms, but
are capable of growing (or expressing their
properties) at 440C.
The presence of these organisms in the water
implies that serious contamination has occurred.

Escherichia coli or ‘Fecal E. coli'
E. coli is regarded as the essential indicator of fecal
pollution of human and animal origin.
It Is defined as a thermotolerant coliforms which
ferment lactose (or mannitol) with production of acid
& gas within 24 h.
Also form indole from tryptophan at this
temperature.
Does not survive in water for long time therefore, is
the best indicator of recent fecal pollution of water.

Fecal streptococci
Are the gram (+), catalase (-), cocci from the GIT of man
and animals.
Occasionally may be found in food & environmental
samples, not known to be contaminated with feces.
Have Lancefield group D Antigen.
Hydrolyze esculin,
Can grow at 450 C in the presence of azide and 40 %
bile.
Can survive at 600C for 30 minutes.

Fecal streptococci cont....
genus Enterococus - E. faecalis & E. faecium being the
most common species in the human faeces.
Those cocci that do not have above mentioned properties
are either S. bovis or S. equinus which are predominant in
ruminants & horses respectively.
 Fecal streptococci (such as the Enterococcus species)
may be present in water samples, but usually only for short
durations.
Hence , if fecal streptococci are present in water sample, it
suggests recent fecal contamination of water.

Sulphite Reducing Clostridia
These are the members of the genus Clostridium.
These bacilli reduce sulphite to sulphide.
The most important organism in the context of water
microbiology is C. perfringens.
Presence of this organisms or their spores, indicate
remote or intermittent fecal contamination.

Pseudomonas aeruginosa
can multiply rapidly in the wide variety of aquatic
environment,
not always found in the human feces & therefore
Is not very good indicator of fecal contamination.
However, it is an important pathogen encountered in
hospital environment and hence
Its detection in water supplies of hospitals and food
establishments has significant value.

Bacteriophage
That infects E. coli can be used as the indicator of fecal
pollution of water.

Collection of water sample
1. For collection,
 use heat sterilised bottles
containing a sufficient volume of
sodium thiosulphate (to neutralise the
bactericidal effect of any chlorine in water.)
 Each bottle of 100 ml capacity
should contain 0.1 ml of fresh 1.8
% (w/v) aqueous solution of
sodium thiosulphate.

Collection of sample from taps
 When collecting the samples from tap,
 Exercise extreme care to avoid contaminating it with
bacteria from the environment.
 Allow water to run for 2 to 3 minutes before collecting it
in the bottle.

Collection of samples from a tap cont….
• In regular use:
Allow the water to run for 2-3 minutes.
• Not in regular use:
Sterilize the tap by heating it either with a blow lamp or
with an ignited piece of cotton soaked in spirit, until it is
unbearably hot to the touch
Cool the tap by allowing the water to run before
sampling.
For collection hold the bottle near the base with one
hand
Remove the stopper & paper cover together & hold in
the fingers
Fill the bottle from a gentle stream of the water from the
tap

Collection of samples from
rivers, lakes or reservoirs
 When sampling from streams or lakes,
 open the bottle at a depth of about 30 cm with its mouth
facing the current.
Eensure that water entering the bottle has not been in
contact with the hand.
if there is no current (e.g. lake), move the bottle
horizontally, so that water flows into it
bring the bottle to the surface, replace the stopper over
it.

Collection of samples from
wells/depth
a heavy bottle with lid having
two cords attached one to the neck & other
to the stopper is used.
lower the bottle to the required depth.
fill it by jerking out the stopper by means of the attached
cord.
raise the bottle quickly to the surface.
replace the stopper.

Transportation & storage of samples
 Collect at least 100ml in each bottle.
 After collection-
label it with full details of the source of the water and
time and date of collection and
deliver it to laboratory as quickly as possible, at least
within 6 hr, keeping it in a cool container and
protected from light.

Counting of indicator organisms
 It is necessary not only to attempt to detect the presence
of the indicator bacteria, but also to innumerate them.
 Greater their number, the greater is the probability of
infection from the supply.
 As the number of indicator bacteria in the water may be
small, large volumes of water have to be cultured.
 Two methods are available for this purpose,
Multiple tube method and
Membrane filtration method.

Counting of indicator organisms
Multiple tube method.
Membrane filtration method

Advantages
 Membrane filtration has advantages over the multiple tube
test
Require less labour and materials and
gives directly quantitative results quickly,
so that any corrective action required to render the
supply safe may be taken sooner.
 Multiple tube methods has the advantages that
 it can show gas formation by the bacteria and
is suitable for the examination of turbid waters
containing small numbers of the indicator bacteria.

Cont...
• Waters containing numerous sapropyhtic
bacteria that might supress the growth of the
coliform.
• Membrane filtration tends to give lower counts
and more false negative results with E. Coli than
the multiple tube methods. (Tillett et al 1988).

Multiple tube test
 Measured volumes (50ml and 10ml) of water and dilution
of water are added to a series of tubes or bottles
containing-
equal volume of double strength MacConkey’s broth
with neutral red.
 Also 1ml of water is added to 5ml of ss medium.
 Media is incubated aerobically at 370C and examined
after 24 hrs.
 The media having the indicator bacteria show growth
with production of acid & gas and characteristic colour
change, (which are absent in those receiving an sample
of water without indicator bacteria.)

 indicated by its colour change to pink with the formation
of acid from lactose in the broth.
An inverted Durham tube is placed in each bottle or
tube of the medium.
Bacteria capable of growth and the production of acid
and gas in MacConkey’s broth are assumed to be
coliform bacilli, i.e, ‘presumptive coliform’.
 From the numbers and distribution of positive and
negative reactions, the most probable number (MPN) of
indicator organism in the sample estimated.
 by reference to statistical tables( McCrady’s table).

Media:
 Mac Conkey broth, double strength:
 Peptone ------------------ 40g
 Sod. Taurocholate------10g
 Lactose--------------------20g
 NaCl-----------------------10g
 Neutral red----------------0.15g,
 D/W------------------------1 litre
 pH: 7.4
distribute in 10ml & 50ml volumes in the tubes with Durham
tube, autoclave at 1150C for 10minutes.
 Mac Conkey broth, Single strength:
dilute DS medium with an equal vol. of D/W
distribute in 5ml vol. in the tubes with Durham tube, autoclave at
1150C for 10 minutes.

Alternative media
 An alternative selective indicator medium is lauryl
tryptose broth.
 In this fermentation of lactose is judged by gas
formation.
 Indole production is to be observed by the addition of
indole reagent after growth.

 Tryptose ---------------------
 Lactose-----------------------
 Sodium chlorode------------
 Dipotassium
hydrogen phosphate--------
 Potassium
dihydrogen phosphate-----
-
 Sodium lauryl sulphate,
D/W---------------------------
40 g
10 g
10 g
5.5 g
5.5 g
0.2 g
1 litre
Lauryl tryptose broth, double strength
(recommended as an alternative to Mac Conkey broth by
American Public Health Association)

Procedure
 For water of good quality:
 1 tube containing 50ml DS medium Aseptically inoculate with corresponding vol.of
 5 tube containing10 ml DS medium well mixed water samples
For water of doubtful quality:
 1 tube containing 50ml DS medium Aseptically inoculate with corresponding vol.of
 5 tube containing10 ml DS medium well mixed water samples.
 5tube containing 5ml SS medium --------- inoculate with 1ml. of well mixed water
samples

Procedure
For water known to be polluted:
5 tube containing10 ml DS medium----- inoculate with
corresponding vol. of water
 5tube containing 5ml SS medium------- inoculate
with 1ml. of well mixed water samples
Prepare 1/10 dil. & higher 10 fold dil. of water in
Ringer’s solution
Two sets of 5 tube containing 5ml SS medium---
inoculate 1 set with 1ml of 1/10 diluted water & other
sets with 1ml of any higher dilution.

Procedure cont...
 Incubate the inoculated media aerobically at 370C.
 After 24h & 48h of incubation, inspect the media.
 Note the no. of cultures of each vol. of water that shows the
production of acid (colour change) & gas (a bubble large
enough to fill the concavity at top of Durham tube).
 These acid and gas producing cultures are considered
‘presumptive positive’ growths of colliform bacilli.
 Cultures not showing both acid & gas at 48h ---- negative.
 Read the probable no. of coliforms per 100ml from the MPN
(most probable number) table of Mac Crady.

Confirmed coliform count: Eijkman test
• Principle:
 some spore bearing bacteria give false positive reaction
in the presumptive coliform test.
 Their presence is most likely to be misleading in the
examination of chlorinated drinking water. (spores are
more resistant to chlorine)
 It is necessary, therefore, to confirm the presence of true
(faecal) coliform bacilli in each vessel showing a
presumptive positive reaction.
 and it may be necessary to determine whether these
coliform bacilli are E. Coli.

 Interpretation:
Gas production in tubes incubated at 370C for 48h----
confirms coliforms.
Gas & indole production in tubes incubated at 440C
for 24h ---- confirms E.coli.
Formation of indole at 44 0C without the formation of
gas from lactose at 44 0C , even if acid is formed-------
-other than E. coli.
For greater assurance, the colonies may be
submitted to oxidase test to exclude the possibility of
Pseudomonas, aeromonas, which gives (+)ve
reaction in this test.
 Refer to the Mac Crady’s probability table & read off
the MPN of confirmed coliform count & confirmed E.
coli per 100ml of water.

Membrane filtration method
 In this method, a measured volume of the water sample
is filtered through a membrane filter( 47mm diameter and
0.45μm pore size) small enough to retain the indicator
bacteria to be counted.
 The membrane is then placed and incubated on a
selective indicator medium, so that the indicator bacteria
grow into colonies on its upper surface.
 These colonies, which are recognised by their colour,
morphology and ability to grow on the selective, are
countered.

Total coliform (presumptive coliform) count by
filtration
For this test the membrane are cultured on pads soaked
with membrane lauryl sulphate broth or an equivalent
medium, which is inhibitory to many non-coliform
bacteria, the coliforms grow as yellow-coloured colonies.
Steps:
1. A measured volumes of well mixed water samples is
filtered through a membrane with a pore size of 0.45µ.
2. The membrane is then placed on a selective indicator
medium (lauryl sulphate medium).
3.Then we incubate it aerobically at 30 0c for 4 hrs followed
by 14 hrs at 37 0c.

4. with chlorinated water, incubate at 25 0c for 6h followed
by 18h at 35 0c.
5. immediately on removal from incubator, count all the
yellow colonies on the surface of membrane irrespective
of size .
6. with reference to volume of water filtered, express the
result as number of presumptive coliforms per 100ml of
samples.

Plate count
It expresses the number of all colony forming bacteria in
1ml of water.
it provides information regarding amount & type of the
organic matter in water.
This may be useful in indicating the efficiency of the
processes used for water treatment.
Also useful in assessing the suitability of water for large
scale production of food and drink.
 Separate plate cultures are incubated at 20-22 0c and at
37 0c.
 At 20-22 0c grows mainly the natural saprophytes of soil
and water.
 At 37 0c grows mainly parasitic bacteria derived from
human and animal excretions.

Classification of the quality of drinking water
based on bacteriological tests
Presumptive
coliform count
per 100 ml
(MPN)
E. Coli count per
100 ml
Class 1
Excellent
0 0
Class 2
Satisfactory
1-3 0
Class 3
suspicious
4-10 0
Class 4
Unsatisfactory
> 10 1 or more

Examination of water

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