CHEMIST

RY
PROJECT
Checking bacterial contamination in drinking water by
testing sulphide ion and also testing the hardness of
water

B.AISHWAR
YA

I.

1.1

INTRODUCTION

Water available these days is highly polluted in urban areas.

Pollutants from paper mill, gas works, sewage works, leather
industry, paints and chemical plants are responsible for water
pollution.
Stagnant water is highly unsafe because it contains lots of
bacteria. Sulphide ions are present in water when anaerobic
bacteria decompose organic matter or reduce sulphates to
sulphides.

1.2 WHAT DOES IMPROVED DRINKING WATER

AND SANITATION MEAN?
Improved drinking-water source
An improved drinking-water source is defined as one that, by
nature of its construction or through active intervention, is
protected from outside contamination, in particular from
contamination with faecal matter.
Improved sanitation facilities
For MDG monitoring, an improved sanitation facility is defined
as one that hygienically separates human excreta from human
contact.
1.3

DRINKING WATER QUALITY

Water quality refers to the chemical, physical, biological,

and radiological characteristics of water. It is a measure of the
condition of water relative to the requirements of one or more
biotic species and or to any human need or purpose. It is most
frequently used by reference to a set of standards against which
compliance can be assessed. The most common standards used
to assess water quality relate to health of ecosystems, safety of
human contact, and drinking water.

1.4 DRINKING WATER QUALITY GUIDELINES

AND STANDARDS
The Bureau of Indian Standards (BIS) has specified drinking
water quality standards in India to provide safe drinking water
to the people. It is necessary that drinking water sources should
be tested regularly to know whether water is meeting the
prescribed standards for drinking or not and, if not, then, the

extent of contamination/ unacceptability and the follow-up

required.
Apart from BIS specification for drinking water, there is one
more guideline for water quality, brought out by Ministry of
Water Resources, Government of India in 2005. This is known
as Uniform Protocol for Water Quality Monitoring. A need has
arisen to have a separate uniform protocol for Drinking Water
Quality Monitoring in view of increasing risk of gynogenic and
anthropogenic contamination.
Keeping in view requirement of preparing Uniform
Drinking Water Quality Monitoring Protocol, the Ministry of
Drinking Water and Sanitation (MDWS), Government of India
constituted an Expert Group which prepared the Protocol. The
Drinking Water Quality Monitoring protocol describes specific
requirements for monitoring drinking water quality with a
view to ensure provision of safe drinking water to the
consumers.
Definition of drinking water quality
BIS has set specifications in IS10500 and subsequently the
revised edition of IS 10500: 2012 in Uniform Drinking Water
Quality Monitoring protocol.
Some parameters apart from those mentioned in IS 10500: 2012
may also be measured if the States deem it necessary. This
standard has two limits i.e. Acceptable limits and permissible
limit in absence of alternate source. If any parameter exceeds
the limit, that water is considered unfit for human
consumption.

S.NO.

TEST
PARAMETER

DRINKING WATER
SCPECIFICATION
REQUIREMEN
T

PERMISSIBLE
LIMIT IN THE
ABSENCE OF
ALTERNATE
SOURCE

1.

ODOUR

AGREEABLE

AGREEABLE

2.

pH VALUE

6.5 - 8.5

AGREEABLE

3.

TURBIDITY

4.

TASTE

AGREEABLE

AGREEABLE

5.

TOTAL
DISSOLVED
SOLIDS(G/L)

500

2000

6.

TOTAL
ALKANITY
(mg/l)

200

600

7.

TOTAL
HARDNESS
(mg/l)

200

600

8.

CALCIUM
(mg/l)

75

200

9.

MAGNESIUM
(mg/l)

30

100

10.

CHLORIDE(mg/
l)

250

1000

11.

RESIDUAL FREE
CHLORINE(mg/
l)

0.2

12.

SULPHATE
(mg/l)

200

400

13.

NITRATE
NITROGEN
(mg/l)

45

NO
RELAXATION

14

FLUORIDE
(mg/l)

1.0

1.5

15

IRON (mg/l)

0.3

NO
RELAXATION

II.

2.1 TESTING FOR MICROBIAL CONTAMINATION

Microbial Contamination Test is conducted on non-sterile
products to check:

 The level of microbial (bacterial and fungal) contamination

Presence/ absence of certain pathogenic microorganism
in order to assure product
safety.

2.2 WHO GUIDELINES FOR MICROBIAL

CONTAMINATION
Securing the microbial safety of drinking-water supplies is
based on the use of multiple barriers, from catchment to
consumer, to prevent the contamination of drinking-water or to
reduce contamination to levels not injurious to health. Safety is
increased if multiple barriers are in place, including protection
of water resources, proper selection and operation of a series of
treatment steps and management of distribution systems
(piped or otherwise) to maintain and protect treated water
quality. The preferred strategy is a management approach that
places the primary emphasis on preventing or reducing the
entry of pathogens into water sources and reducing reliance on
treatment processes for removal of pathogens. In general terms,
the greatest microbial risks are associated with ingestion of
water that is contaminated with human or animal (including
bird) faeces. Faeces can be a source of pathogenic bacteria,
viruses, protozoa and helminths.

2.3 BACTERIA AND CONTAMINATION

Both bacteria and viruses are microorganisms regulated by
EPAs Maximum Contaminant Levels (MCLs) criteria. Viruses
are the smallest form of microorganisms capable of causing
disease, particularly those of a faecal origin infectious to
humans by waterborne transmission; bacteria are typically
single-celled microorganisms that can also cause health
problems in humans, animals or plants, despite many forms
ability to aid in water pollution control.
COMMON TYPES OF BACTERIA AND VIRUSES
Various types of bacteria/viruses are categorized as pathogens,
disease-causing organisms that can be found in pre-treated
and/or inadequately treated water. Here is a list of EPA
regulated bacteria/viruses in drinking water, and their health
risks:
Legionella, a bacteria found naturally in the environment
typically in water, thrives in warm waters; this bacteria
in water is a health risk if aerosolized (e.g., in a shower or
air conditioning system) and inhaled, resulting in a type of
pneumonia known as Legionnaires disease.
Enteroviruses are small viruses, such as polioviruses,
echoviruses and coxsackieviruses, living in the intestines
of infected humans or animals; in addition to the three
different polioviruses are 62-nonpolio enteroviruses that
can cause disease in humans ranging from gastroenteritis
to meningitis.

2.4

INFECTIVE DOSE

Waterborne infections The pathogens that may be transmitted

through contaminated drinking-water are diverse in
characteristics, behaviour and resistance. Table 7.1 provides
general information on pathogens that are of relevance for
drinking-water supply management. Waterborne transmission
of the pathogens listed has been confirmed by epidemic- logical
studies and case histories. Part of the demonstration of
pathogenicity involves reproducing the disease in suitable
hosts. Experimental studies in which healthy adult volunteers
are exposed to known numbers of pathogens provide
information, but these data are applicable to only a part of the
exposed population; extrapolation to more vulnerable
subpopulations is an issue that remains to be studied in more
detail.

2.5 CULTURE MEDIA

2.5.1 NUTRIENT PADS
Nutrient pad sets for colony counting by the membrane filter
method. These nutrient pad sets are dehydrated culture media
that are already sterilised and individually inserted in Petri

dishes. The special feature of this alternative to agar media is

the long shelf life of 24 month by storage at room temperature.
All nutrient pad set types are supplied
with the appropriate membrane filters
(pore size and colour), which are also
presterilized and individually packaged.
The membrane filters are customized to
meet the requirements of microbial
detection, and are supplied in 47- or 50mm diameters.

2.5.2 BROTHS
The two main types of bacterial growth media used are liquid
broth and solid, Jell-o-like agar. Each has specific advantages
and disadvantages. The growing environment used will
depend on what the researcher wants to do with, or learn from,
the microbes.

Nutrient Broth Bacterial Growth Medium

Nutrient broth is typically made of a powdered beef extract that
contains peptones (broken down proteins). The powder is
dissolved in water, put in test tubes, and sterilized.

Broth is convenient, as most bacteria will grown in this type of

medium, even those with widely different aero tolerances
(oxygen requirements).

2.6 TEST METHODS

2.6.1 PRESENCE-ABSENCE
The Presence-Absence (P-A) test is a presumptive detection for
coliforms in water. The test is a simple modification of the
multiple-tube procedure.1 One 100 mL test sample is
inoculated into a single culture bottle to obtain qualitative
information on the presence or absence of coliforms, through
the presence or absence of lactose fermentation.1 This test is
based on the principle that coliforms and other pollution
indicator organisms should not be present in a 100 mL water
sample.2-4
Comparative studies with the membrane filter procedure
indicate the P-A test may maximize coliform detection in
samples containing many organisms that could overgrow
coliform colonies and cause problems in detection.1 The P-A
test is described in standard methods for water testing1 and
U.S. EPA.
PROCEDURE
The nitrogen, vitamin, and amino acids sources are provided by
Enzymatic Digest of Gelatine, Enzymatic Digest of Casein, and
Beef Extract. Lactose is the fermentable carbohydrate.
Dipotassium Phosphate and Monopotassium Phosphate
provide buffering capacity. Sodium Chloride maintains the
osmotic balance of the medium. Sodium Lauryl Sulphate is the
selective agent, inhibiting many organisms except coliforms.

Bromcresol Purple is used as an indicator dye; lactosefermenting organisms turn the medium from purple to yellow
with or without gas production.

2.6.2 MOST PROBABLE NUMBER

The most probable number method, otherwise known as the
method of Poisson zeroes, is a method of getting quantitative
data on concentrations of discrete items from positive/negative
(incidence) data.
There are many discrete entities that are easily detected but
difficult to count. Any sort of amplification reaction
or catalysis reaction obliterates easy quantification but allows
presence to be detected very sensitively. Common examples
include microorganism growth, enzyme action, or catalytic
chemistry. The MPN method involves taking the original
solution or sample, and subdividing it by orders of magnitude
(frequently 10 or 2), and assessing presence/absence in
multiple subdivisions.
The degree of dilution at which absence begins to appear
indicates that the items have been diluted so much that there
are many subsamples in which none appear. A suite of
replicates at any given concentration allow finer resolution, to
use the number of positive and negative samples to estimate
the original concentration within the appropriate order of
magnitude.

2.6.3 MEMBRANE FILTRATION MENTHOD

The Membrane Filter (MF) Technique was introduced in the

late 1950s as an alternative to the Most Probable Number
(MPN) procedure for microbiological analysis of water
samples. The MF Technique offers the advantage of isolating
discrete colonies of bacteria, whereas the MPN procedure only
indicates the presence or absence of an approximate number or
organisms (indicated by turbidity in test tubes).
The MF Technique was accepted by the U.S. EPA for
microbiological testing of potable water in the 11th edition
of Standard Methods for the Examination of Water and Wastewater.
In the 1978 publication, Microbiological Methods for Monitoring
the Environment, the U.S. EPA stated that the MF Technique is
preferred for water testing because it permits analysis of larger
samples in less time.
ADVANTAGES OF MF TECHNIQUE

Permits testing of large sample volumes.

Reduces preparation time as compared to many

traditional methods.

Allows isolation and enumeration of discrete colonies of

bacteria.

Provides presence or absence information within 24 hours.

Effective and acceptable technique. Used to monitor

drinking water in government laboratories.

Useful for bacterial monitoring in the pharmaceutical,

cosmetics, electronics, and food and beverage industries.

Allows for removal of bacteriostatic or cidal agents that

would not be removed in Pour Plate, Spread Plate, or MPN
techniques.
III.

EXPERIMENT
AIM : To test the contamination of water by bacteria by
checking the sulphide ions concentration and find out cause of
contamination.

MATERIALS REQUIRED :
Cadmium acetate 50g
Zinc acetate 50g
Distilled water 500mL
Iodine solution 0.025M
Conc. HCl
Na2 S2 O3 0.05M
Starch solution as indicator.

THEORY:
Sulphide ions are readily oxidised therefore, care should be
taken at the time of sampling to exclude air by flushing it with
nitrogen or carbon dioxide. This is a difficult process. The best
way is to 'fix' the sample immediately after solution.

PROCEDURE:

 Take 50g of cadmium acetate and 50g and 50g of zinc

acetate and dissolve in water.
Neutralize the solution with a little excess of alkali.
Take 20mL of cadmium-zinc acetate solution and add 80
mL of sample of given water to obtain a total volume of
about 100 mL.
Take 100 mL of fixed sample solution in titration flask.
Add 20 mL of 0.025 M iodine solution.
Add immediately 15mL of 50% of HCl solution in water.
Add starch solution as indicator.
Titrate the excess of iodine against 0.05 M Na2S2O3 .
Calculate the amount of S-2 ions in the original samples
from the amount of iodine used in reaction with H2S.
Repeat the same procedure with other samples of water.

ENDPOINT :
Blue to colourless.

CHEMICAL REACTION :
I2 + H2S = 2HI + S
I2 + 2Na2S2O3 = 2I- + Na2S4O6 + 2Na+