BACTERIOLOGY OF WATER

• Safe and wholesome water is defined as water that is

free from pathogenic agents, free from harmful
chemical substances, pleasant to the taste and usable
for domestic purposes.
• Many major human diseases, for example, typhoid fever,
cholera and other diarrheal diseases, poliomyelitis and
viral hepatitis A and E are waterborne.
• Both chemical and bacteriological examination of water
supply from the source to the consumer should be
regularily and systematically done, though in several
cases chemists and bacteriologists may disagree
 The aim of microbiological examination of water supplies is to
detect whether pollution of the water by pathogenic organisms has
occurred or not.
 It is impracticable to attempt directly to detect the presence of all
the different kinds of water-borne pathogens, any of which may be
present only intermittently.
 Instead, reliance is placed on testing the supply for microorganisms
which indicate that fecal pollution has taken place.
 • Natural water bacteria: This group includes those
organisms that are commonly found in water free
from gross pollution.
BACTERIAL • Soil bacteria: These organisms are frequently
washed into water during heavy rains and are not
FLORA IN nor mal inhabitants of water.
• Sewage bacteria: In this group many of the

WATER bacteria are normal inhabitants of the intes tine of

man and animals. Others live mainly on
decomposing organic matter of either ani mal or
vegetable origin
 FACTORS DETERMINING THE NUMBER OF BACTERIA IN WATER

 Surface or deep water: Surface water is more likely to be contaminated

 Salinity: In saline water number of bacteria is less as compared to fresh water.
However, halophilic bacteria can survive in saline water.
 Mineral springs: These are usually pure and most of the organisms found in water
derived from them come from imperfectly sterilized bottles.
 Nutrition: When organic matter is plentiful, organisms are abundant, when it is
scarce they are few, and tend to die out.
 Temperature: When nutrition is available, rise in temperature leads to
multiplication, oth erwise, the number decreases. Low temper ature favors
survival of bacteria.
 Light: Day light with the wavelength of 300 -400 nm is bactericidal.
 Acidity: Acidity of water has a bactericidal action, thus purifying water.
 Dissolved oxygen: It is essential for survival of aerobes.
 Protozoal content: Certain flagellates exterminate bacteria in water
and bring down their number.
 Rain: Early rain washes large number of bacteria from the soil which
may contaminate water sources. Subsequent rains dilute the bacterial
population.
 Storage: Storage of water decreases bacterial count due to
sedimentation and devitalization.
 INDICATOR ORGANISMS

 Microorganisms for use as indicators of fecal pollution should satisfy several criteria.
 They should be present in feces in greater numbers than any 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
environ ment and disinfection processes.
 Usually a number of indicator organisms are sought. Such as coliforms and E. coli other
bacteria also sometimes used as indicators of fecal pollution such as streptococcus
fecalis and clostridium perfringers.

Coliforms are bacteria that are always present in the digestive tracts of animals,
including humans, and are found in their wastes
COLLECTION OF WATER SAMPLES

 For collection, use heat-sterilized bottles con taining a sufficient

volume of sodium thiosulphate to neutralize the bactericidal
effect of any chlorine or chloramine in the water which may lower
bacterial counts by continued activity.
 Each bottle of 100 ml capacity should contain 0.1 ml of a fresh 1.8
percent (w/v) aqueous solution of sodium thiosulphate
1. Sampling from a tap or pump outlet: When collecting the sample from taps,
exercise extreme care to avoid contaminating it with bacteria from the
environment. Allow water to run to waste for 2-3 min before running it into the
bottle.
2. Sampling from reservoir (streams, rivers, lakes and tanks): When sampling from
streams or lakes, open the bottle at a depth of about 30 cm with its mouth facing
the current and ensure that water entering the bottle has not been in contact
with the hand. Collect at least 100 ml in each bottle.
3. Sampling from a dug well: A stone of suit able size is tied with the bottle. Then a
clean cord of suitable length is tied with the bottle and lowered into the well.
Immerse the bottle completely in the water. When the bottle is filled, pull it out,
stopper it and wrap it in a kraft paper. The bottle should not touch the sides of the
well any time
 • label it with full details of the
Transport source of the water and time and
Stopper date of collection, and deliver it
to the laboratory as quickly as
the possible, at least within 6 hours,
keeping it in a cool container and
bottle protected from light.
 BACTERIOLOGICAL EXAMINATION OF WATER

The following tests are generally done for routine bacteriological analysis of water:
A. Plate count
B. Counting of indicator organisms
a. Multiple tube test
1. Total coliform count or Presumptive coliform count
2. Eijkman test: Fecal coliform and confirmed Escherichia coli count.
3. Count of fecal streptococci
4. Count of Clostridium perfringens
b. Membrane filtration tests.
 • The plate count expresses the number of all
colony forming bacteria in 1 ml water. It is of
limited value by itself, but as a supplementary
Plate test it provides information about the amount
and type of organic matter in the water which
Count may be useful in indicating the efficiency of
the processes used for water treatment or the
suitability of the water for large-scale
production of food and drink.
 • As the number of indicator
B. bacteria in the water may be
Counting small, large volumes of the water
have to be cultured.
of • Two methods are available for
Indicator this purpose, the multiple tube
method and the membrane
Organisms filtration met
 a. Multiple Tube Test

 Measured volumes of water and dilutions of water are added to a

series of tubes or bottles containing a liquid indicator growth medium.
 The media receiving one or more of the indicator bacteria show growth
and a characteristic color change which are absent in those receiving an
inoculum of water without indicator bacteria.
 From the number and distribution of positive and negative reactions,
the most probable number (MPN) of indicator organisms in the sample
may be estimated by reference to statistical tab
Advantages
The multiple tube method 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, e.g. waters containing numerous
saprophytic bacteria that might suppress growth of the
coliforms.
 1. Presumptive Coliform Count (Total Coliform Count)
The test is called presumptive because the reaction observed
may occasionally be due to the presence of some other
organisms and the presumption that the reaction is due to
coliform organisms has to be confirmed
Indicator Medium

 The indicator medium used most has been MacConkey broth containing
bromocresol purple to indicate by its color change to yellow the
formation of acid from the 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 broth are assumed to be coliform bacilli, i.e. ‘presumptive
coliforms’.
 The probable number of coliforms per 100 ml are read off from the probability tables
of McCrady. This is known as the ‘presumptive coliform count’ or the most probable
number of coliforms (MPN).
2. Eijkman Test :
 Fecal Coliform and Confirmed Escherichia Coli Count Some spore-
bearing bacteria give false-positive reactions in the presumptive
coliform test.
 It is necessary, therefore, to confirm the presence of true (‘fecal’)
coliform bacilli.
 The Eijkman test : is usually employed to find out whether the coliform bacilli
detected in the presumptive test are E. coli.
 After the usual presumptive test, subcultures are made from all the tubes/ bottles
showing acid and gas to fresh tubes of single strength MacConkey’s medium already
warmed to 44°C.
 Incubation at 44°C should be carried out in thermostatically controlled water baths
that do not deviate more than 0.5°C from 44°C.
 Those showing gas in Durham’s tubes contain E coli. Further confirmation of the
presence of E. coli can be obtained by testing for indole production and citrate
utilization
 Count of Fecal Streptococci

 If there is difficulty in interpreting the results of the presumptive

coliform and confirmed E. coli tests, as when presumptive coliforms
are present but E. coli is absent, a demonstration of the presence of
fecal streptococci will confirm the fecal origin of the coliform bacilli.
 Subcultures are made from all the positive bottles in the presumptive
coliform test into tubes containing 5 ml of glucose azide broth.
 The presence of Enterococcus. fecalis is indicated by the
production of acid in the medium within 18 hours at 45°C.
 The positive tubes should be plated onto MacConkey’s agar for
confirmation.
 Millipore membrane technique can also be adopted for this
purpose.
 Count of Clostridium Perfringens

 This is tested by incubating varying quantities of the water in litmus milk medium
(anaerobically) at 37°C for five days and looking for stormy fermentation.
 Membrane Filtration Tests

 In this method, a measured volume of the water sample is filtered

through a membrane with a pore size small enough to retain the
indicator bacteria to be counted on its surface.
 The membrane is then placed and incubated on a selective indicator
medium at the appropriate temperature, so that the indicator bacteria
grow into colonies on its upper surface.
 These colonies, which are recognized by their color, morphology and
ability to grow on the selective medium, are counted and the
bacteriological content of water calculated.
 Tests for Pathogenic Bacteria

 Specific pathogens such as typhoid bacilli or cholera vibrios may have to be

looked for in water by employing enrichment and selective media under
special circumstances.
 This used to be done by adding the water samples to tenfold concentrated
liquid media, incubating and subculturing onto appropriate solid media

 Isolation of S. Typhi: For isolation of S. Typhi, equal volume of water is added to

double strength selenite broth followed by incubation and subculture on Wilson
and Blair’s medium
Isolation of V. cholerae: For isolation of V. cholerae, alkaline peptone water
(l0x ) is mixed with nine times its volume of water, incubated and subcultured on bile
salt agar.
 A simpler and more sensitive method is to filter the water sample through membrane
filters and incubate the filters on appropriate solid media.
 Viruses in Water

 It is recommended that, to be acceptable, drinking-water should be free

from any viruses infections for man.
 Methods are available for the isolation of enteroviruses and other
cytopathogenic viruses from water but they do not form part of routine
testing.
 As a general rule, it is assumed that the viruses in water are destroyed by
chlorination, when the concentration of free residual chlorine is at least 0.5
mg per liter, for a minimum contact period of 30 minutes at pH below 8 and
a turbidity of 1 nephalometric turbidity unit or less.
 Protozoa in Water

 Species of protozoa known to have been transmitted by the ingestion of

contaminated drinking-water include Entamoeba histolytica, Giardia
spp. and rarely, Balantidium coli.
 However, there is no good indicator for protozoal contamination of
water.
 Coliform counts are not reliable as indicators of protozoal
contamination of chlorinated water as they are more resistant to
chlorine than are coliforms
BACTERIOLOGY OF MILK
Human infections may be caused by the ingestion of
animal milk which contains microorganisms derived
either from the animal, e.g. by contamination with its
feces, or from the environment or from milk handlers
such as dairy workers.
 Bacteriological Examination of Milk

• This is estimated by doing plate

counts with serial dilutions of the
1. Viable milk sample.
• Raw milk always contains
count bacteria, varying in number from
about 500 to several million per
ml
 2. Test
• This is tested by inoculating varying dilutions of milk
into 3 tubes of MacConkey’s fluid medium with
Durham tube and noting the production of acid and

for gas after incubation at 37°C for 48 hours.

• Contamination with coliforms comes mainly from
dust, dirty utensils and dairy workers.
coliform • All coliforms are killed by adequate pasteurization
and their presence in pasteurized milk indicates

bacilli faults in pasteurizer or postpasteurization

contamination
 • It depends on the reduction of methylene blue by
3. Methylene bacteria in milk when incubated at 37°C in complete
darkness. The rate of reduction is related to the degree
blue of bacterial contamination. Raw milk is considered
satisfactory if it fails to reduce the dye in 30 minutes
reduction test under standard conditions.

• The test is performed by adding I ml of standard

methylene blue solution to 10 ml of milk in a test tube.
Procedure The tube is incubated in the dark at 37°C. The milk is
considered satisfactory, if it fails to reduce the dye in 30
minutes.