DETERMINATION OF TC AND FC OF WATER

ENVIRONMENTAL ENGINEERING-II SESSIONAL

CE-4402

SUBMITTED BY

SHUVA CHANDRA BOSE

Student ID: 161116
SECTION: B

SUBMITTED TO

DR. MD. AKRAMUL ALAM

PROF. CE, DUET
SHAHAB UDDIN
ASSISTANT PROF. CE, DUET

DEPARTMENT OF CIVIL ENGINEERING

DHAKA UNIVERSITY OF ENGINEERING AND TECHNOLOGY, GAZIPUR

Page 1 of 6
1.0 INTRODUCTION
A variety of different microorganisms are found in untreated water. Most of these organisms
do not pose a health hazard to humans. Certain organisms, referred to as pathogens, cause
disease to humans which include species of bacteria, viruses and protozoa. These organisms
are not native to aquatic systems and usually require an animal host for growth and
reproduction. They can, however, be transported by natural water systems, thus becoming a
temporary member of the aquatic community. Many species of pathogens arc able to survive
in water and maintain their infectious capabilities for significant periods of time. From the
perspective of human use and consumption, the most important organisms in water are the
pathogens.

Total coliform (TC) = Fecal coliform (FC) + Non-fecal coliform.

Fecal coliforms (thermo-tolerant coliform) are a subgroup of total coliforms, which live in the
warm blooded animals and have the same properties as the total coliforms~but tolerate and
grow at the higher selective temperature range of 44°C to 44.5 °C.

1.1 OBJECTIVE
The main objective is to determine Microbiological Quality of Water: -
(1) To Determine the Total Coliforms (TC)
(2) To Determine the Fecal Coliforms (FC)

2.0 MATERIALS AND METHODOLOGY

2.1 Apparatus
(a) incubator,
(b) rack for incubation of Petri-dishes,
(c) filter funnel, 100 ml capacity,
(d) porous support for filter,
(e) filler support,
(f) suction vessel,
(g) syringe with two way valve for providing the vacuum needed for the filtration,
(h) sample vessel,
(i) forceps,
(j) bottle containing alcohol,
(k) bottle containing sterile buffer,
(1) plastic Petri-dishes,
(m) pipettes.
By way of example, the procedures for preparing small quantities of media are given below
for M-Endo MF broth and MFC broth:
(1) M-Endo MF broth for TC:
 Dissolve 2.4 gm of dehydrated culture medium in 50 ml of distilled water and add
1 ml of 95% ethyl alcohol.
 Sterilize by heating gently just to the boiling point.

Page 2 of 6
 The medium can be stored for up to 4 days in the refrigerator. About 50 ml of medium is
sufficient for some 25 tests.
(2)MFC broth for FC:
 Dissolve 1.9 gm of the dehydrated medium in 50 ml of distilled water containing
0.5 ml of 1.0% rosolic acid in 0.2 mol/l sodium hydroxide solution.
 Heat the medium to boiling point.
 Rapidly remove from the heat and cool to below 45 °C.
The prepared medium should not be sterilized by autoclaving. It can be stored for up to 4
days in a refrigerator.

2.2 Methodology

Membrane Filter Method

Volume of Water Sample for Filtration: Since the filtration area is relatively small, it can
only support the growth of a limited number of colonies. The optimum number is between
20 and 80 colonies, with a maximum of 200. If this figure is exceeded, very small colonies
or superimposed colonies may develop, or growth inhibition due to overpopulation may
result. The choice of the volume of sample to be filtered will depend on the type of water.
As a general rule, the following filtration volumes (Table 3) should be employed.

3 Table 3: Volume of water to be filtered for various types of water

Type of water Volume of sample to be filtered (ml)
Good quality treated 50-100
Untreated drinking water 10-50
Surface water 1-10
If the origin of the sample is unknown and its probable bacterial content is uncertain, water
volumes differing by a factor often should be filtered in order to find the appropriate range
for the analysis. If the volume to be filtered is less than 10 ml, at least 20 ml of sterile
dilution water should be placed in the funnel prior to the filtration.

Culture Media: Various media can be used for the examination of coliform organisms by
the membrane-filtration method. Of these, lactose tergitol agar, lactose TTC, tergitol agar
and lauryl sulfate lactose broth may be used for coliform organisms at 35 °C or 37 °C, and
for fecal coliform organisms at 44 °C. Endo-type media should only be used for coliform
counts at 35 °C or 37 °C, and MFC broth at 44°C for fecal coliform counts. Although all these
media rely on the fermentation of lactose for the detection of presumptive coliform
organisms, the characteristic reaction varies with each medium. The characteristic metallic
sheen of colonies on Endo-type media depends on the formation of aldehyde.
Although it is possible to prepare the media from the basic ingredients, this may be
impractical for a small laboratory. The use of dehydrated media is therefore
recommended. The media can be prepared as a broth, and used together with nutrient
absorption pads, or as solid agar plates.
This section describes the general procedures. It should be noted that different types of
filtration units and equipment are available in the market for performing the tests.
Page 3 of 6
 Determination of Total Coliforms (TC) :

(A) Connect the Erlenmeyer (side-arm) flask to the vacuum source (turned off) and place
the porous support in position. If an electric pump is used, it is advisable to put a second
flask between the Erlenmeyer and the vacuum source; this second flask acts as a water
trap and thus protects the electric pump.
(B) Open a Petri-dish and place a pad in it.
(C) With a sterile pipette add 2 ml of selective broth medium to saturate the pad.
(D) Assemble the filtration unit by placing sterile membrane filter on the porous support,
using forceps sterilized earlier by flaming.
(E) Place the upper container in position and secure it with the special clamps. The type of
clamping to be used will depend on the type of equipment
(F) Pour the volume of sample chosen as optimal, in accordance with the type of water, into
the upper container. If the test sample is less than 10 ml, at least 20 ml of sterile dilution
water should be added to the top container before filtration applying the vacuum.
(G) After the sample has passed through the filter, disconnect the vacuum and rinse the
container with 20-30 ml of sterile dilution water. Repeat the rinsing after all the water
from the first rinse has passed through the filter.
(H) Take the filtration unit apart and using the forceps, place the membrane filter in the
Petri-dish on the pad with the grid side up. Make sure that no air bubbles are trapped
between the pad and the filter.
(I) Invert the Petri-dish for incubation.
(J) Incubate at 35 °C or 37 °C for 18-24 hours with 100% humidity (to ensure this, place a
piece of wet cotton wool in the incubator). If ointment containers or plastic dishes with
tight-fitting lids are used, humidification is not necessary.
Colonies of coliform bacteria are a medium red or dark red color, with a greenish gold or
metallic surface sheen. This sheen may cover the entire colony or appear only in the centre of
the colony. Colonies of other types should not be counted. The colonies can be counted with
the aid of a lens.

Determination of Fecal Coliforms (FC):

The procedure for fecal coliforms is similar to that used for determining total coliforms. Filter
the sample as described, and place the membrane filler on the pad saturated with, for example,
MFC medium.

(A)Place the dishes in an incubator at 44±0.5 °C for 24 hours at 100% humidity.

Alternatively, tight-fitting or sealed Petri-dishes may be placed in water-proof plastic
bags for incubation.
(B) Submerge the bags in a water-bath maintained at 44±0.5°C for 24 hours. The plastic
bags must be below the surface of the water throughout the incubation period. They
can be held down by means of a suitable weight, e.g., a metal rack.
Colonies of fecal coliform bacteria are blue in color. This color may cover the entire colony,
or appear only in the center of the colony. Colonies of other types should not be counted. The
colonies can be counted with the aid of a lens.

Page 4 of 6
 3.0 DATA

The colonies for total coliforms (TC) are obtained with the aid of a lens 190
The colonies for fecal coliforms (FC) are obtained with the aid of a lens 70

CALCULATION

Total coliform per 100 ml = (190) x (100) / (100) = 190 CFU

Fecal coliform per 100 ml = (70) x (100) / (100) = 70 CFU

3.0 RESULTS AND DISCUSSION

3.1 Results
The Total coliform per 100 ml from this experiment has been found 190 CFU/100ml and
The Fecal coliform per 100 ml from this experiment has been found 70 CFU/100ml

3.2 Discussion
This experiment was carried out to determine the TC and FC of water. After completing this
experiment TC and FC of water was found . The Total coliform per 100 ml from this experiment
has been found 190 CFU/100ml and The Fecal coliform per 100 ml from this experiment has been
found 70 CFU/100ml. But according to ECR (1997) drinking water standard is zero and it is easy
seen that the sample water was not prefer for drinking water standard .

4.0 CONCLUTION AND RECOMMENDATION

This drinking water sample was analyzed for the presence of total coliform. Since it is too
costly to test water samples for every possible organism that may cause disease, the coliform
group of bacteria are used as an indicator of water quality and to determine suitability for
drinking water purposes. Coliforms are a group of bacteria found in the intestines of humans
and other animals. Coliforms also occur naturally in the environment, including in soil, on
Page 5 of 6
vegetation and in surface waters such as lakes, rivers and streams. Most members of the
coliform group do not cause disease. When found in drinking water, coliform bacteria indicate
that contamination of the drinking water supply has occurred, and that other disease-causing
bacteria could also get into the water supply. The safety of a water supply depends upon
proper construction and protection against contamination. A favorable bacteriological
analysis alone should not be accepted as conclusive evidence of the safety of a water supply.

References

[1] Engineering School UCD (1972). Course Notes - Water Quality Management.
Separation of Suspended Solids 5 - Filtration.

[2] CE 332 Environmental Engineering- Lab I (Lab Manual).

Page 6 of 6