1. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
INTERNATIONAL JOURNAL OF BIOTECHNOLOGY
(IJBT)
ISSN: (Print)
ISSN: (Online) IJBT
Volume 1, Issue 1, January- March (2012), pp. 01-07 ©IAEME
© IAEME: www.iaeme.com/ijbt.html
A STUDY ON THE PRESENCE OF FECAL POLLUTION
INDICATOR BACTERIA IN MUTTUKADU BACK
WATERS, EAST COAST OF TAMIL NADU
C.Ganga Baheerathi1, Research scholar, Sathyabama University
K .Revathi2, Ethiraj College for women, Chennai, India
ABSTRACT
Coliform bacteria are a collection of relatively harmless microorganisms that live
in large numbers in the intestines of man, warm and cold-blooded animals. A specific
subgroup of this collection is the fecal coliform bacteria, the most common member
being Escherichia coli. The presence of fecal coliform bacteria in aquatic environments
indicates that the water has been contaminated with the fecal material of man or other
animals. The presence of fecal contamination is an indicator that a potential health risk
exists for individuals exposed to this water. The current study is aimed at analyzing the
presence of fecal coliform in Muttukadu back waters, where different kinds of fishes,
prawns and shell fishes are harvested for human consumption. The water samples were
collected in pre–monsoon, monsoon and post-monsoon seasons. Water analysis to find
the presence of fecal indicator was done by multiple tube tests to assess the MPN values.
The presence of Escherichia coli is further confirmed by membrane filtration test and
biochemical tests. Analysis showed higher number of fecal indicator in monsoon,
followed by post monsoon seasons. The number was comparatively less in pre monsoon
season
INTRODUCTION
Water quality is a vital aspect for the survival and well being of the living
resources, especially in the coastal and estuarine areas. Some of these areas are now
under the direct threat from the increasing load of various pollutants. In order to quantify
and understand the relationship of pollution indicator and human pathogenic bacteria
with environmental factors, several investigators have examined distribution of these
1
Department of Micro Biology, Ethiraj College for Women, Egmore, Chennai
2
Department of Zoology, Ethiraj College for Women, Egmore, Chennai
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2. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
groups of bacteria and certain viruses in coastal waters (Ramaiah and Chandramohan
1993 ; Ruiz et al .2000 ; Ramaiah and De 2003 ). Land drainages, domestic sewage
outfalls, and other discharges alter the abundance and type of both autochthonous and
allochthonous microbial populations in the near shore environments.Members of two
bacteria groups, coliforms and fecal streptococci, are used as indicators of possible
sewage contamination because they are commonly found in human and animal feces.
Although they are generally not harmful themselves, they indicate the possible presence
of pathogenic bacteria, viruses, and protozoan that live in human and animal digestive
systems. Therefore, their presence in streams suggests that fecal contamination has
occurred and other human pathogenic microorganisms might also be present. Swimming
and eating shellfish from the water might be a health risk.
Since it is difficult, time-consuming, and expensive to test directly for the
presence of a large variety of pathogens, water is usually tested for total coliforms and
fecal coliforms. Faecal coliform bacteria have been widely used as indicators of water
contamination by the fecal material of humans and other warm-blooded animals (APHA
1992;Bordalo 1993)Basically water analysis is done for seeing the presence of fecal
indicator bacteria which might indicate the presence of other human pathogenic bacterial
population of intestinal origin. Marine bivalves accumulate large number of bacteria from
the immediate environment due to its filter feeding nature. (Jan A. Olafsen et al .1993)
Detection and enumeration of indicator organisms are of primary importance for the
monitoring of sanitary and microbiological quality of water (Gunnison, 1999). Total
Coliform and Fecal Coliform counts are the most widely used bacteriological procedures
for assessment of the quality of drinking and surface waters. Microbiologists rely on the
principle that higher the incidence of sewage indicator bacteria in any environment,
higher would be the chances for human pathogenic bacteria to be present (Brock et
al.1994: Fujioka 2002)
The present study is made in Muttukadu back waters. The Muttukadu backwater
(12°47’N, 80°15’E) is located 36 km from Chennai city, runs parallel to the east coast of
India and opens into the Bay of Bengal, from where lot of fishes, shellfishes are
harvested and the area is surrounded by many aqua cultural farms. Sewage and industrial
effluents from the surrounding area have a greater impact on the Muttukadu back water
affecting the aquatic animals and in turn the human population. These wastes carry
enormous number of microbial pathogens and other heavy metals resulting in greater
economic loss.
MATERIALS AND METHODS
Sampling was done during pre-monsoon, monsoon and post monsoon seasons of
2010. The water samples were collected in sterile screw capped bottles for water quality
assessment. The water sample was brought to the laboratory in portable icebox with in 2
hours.
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3. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
MULTIPLE TUBE TEST
The presence of faecal coliforms aerogenic Escherichia coli in water is
determined by means of the multiple tube test procedure
Presumptive test
Most probable number test was done with three dilutions of the sample. The
dilutions used were 10ml, 1 ml and 0.1ml. Each dilution requires five Macconky broth
media tubes, thus the sample is inoculated in five replicas of each dilution.. First set of
5 tubes with double strength Macconky broth was inoculated with
1 0 . 0 m l o f t h e sample to be tested. Second set of another 5 tubes with
single strength medium with 1.0 ml of the sample and the third set of 5 tubes
with 0.1 ml of the sample using sterile pipettes. After 24-48 hours of incubation at
37c ,the results were noted based on acid production and/or gas production in the tubes.
Acid production during the fermentation was noted by the change in the dye color
indicating the pH change. Small tubes, called Durham’s tubes, were used to collect the
gas bubbles formed during the fermentation. The medium in the inverted Durham's tubes
within the test tubes are replaced by the gas produced, thus enabling the observation. The
values were compared with MPN standard chart. The presence of fecal indicator
organism Echerichia Coli is also checked by inoculating in to two tubes of brilliant green
lactose bile broth. One tube was inoculated at 44.5º c and another tube was incubated at
37º c for 24 hrs.
Confirmed test
The presence of fecal indicator Coliform, Escherichia coli is further confirmed by
streaking in Eosin methylene blue agar from the Positive presumptive tube which is
followed by completed test.
Completed test
The isolated bacteria from EMB were taken to nutrient agar slant and Lactose
broth with Durham’s tube for acid and gas production .Gram’s staining was done with the
growth on the nutrient agar slant.
MEMBRANE FILTRATION TECHNIQUE
One of the easy and fast methods for the analysis of water quality is by using
Membrane filtration technique.100 ml of the sample is passed through a 47 mm
membrane with a pore size of 0.45 µm using a filter funnel and a vacuum system. Any
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4. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
organisms in the sample are concentrated on the surface of the membrane. The filter is
then placed in a Petri dish with Eosin methylene blue agar medium.Appearance of
nucleated colony with metallic sheen confirms the presence of Escherichia coli. Further
biochemical test are done to confirm the presence of E. coli.
RESULTS AND DISCUSSION
TABLE: 1 MOST PROBABLE NUMBER – TABLE: 2 MOST PROBABLE NUMBER
WATER SAMPLE – WATER SAMPLE
PRE – MONSOON SEASON MONSOON SEASON
S.no Sample volume S.no Sample volume
10 ml 1 ml 0.1 ml 10 ml 1 ml 0.1 ml
1 + + + 1 + + +
2 + - - 2 + + -
3 + + + 3 + + -
4 + + - 4 + + +
5 + + + 5 + + +
MPN MPN
values 543= 275* values 553= 900*
Most propable number of Coliforms in 100 ml of Most propable number of Coliforms in 100 ml of
water. (+ ) Acid and Gas produced , ( -) No Acid and water. (+ ) Acid and Gas produced , ( -) No
Gas produced Acid and Gas produced
TABLE: 3 MOST PROBABLE NUMBER – A CAMPARISON OF MPN VALUES OF
WATER SAMPLE WATER TAKEN IN THREE SEASONS
POST – MONSOON SEASON
S.no Sample volume
10 ml 1 ml 0.1 ml
1 + + -
2 + + +
3 + + -
4 + + +
5 + + -
MPN
values 552= 550*
Most propable number of Coliforms in 100 ml of
water. (+ ) Acid and Gas produced , ( -) No Acid and
Gas produced
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5. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
The MPN values are observed and tabulated in Table 1,2 and 3 respectively.
Confirmed test revealed the presence of Escherichia coli with nucleated colonies and
metallic sheen in Eosin methylene blue agar. Eosin methylene blue contains the dye
methylene blue, which inhibits the growth of gram – positive organisms. In the
presence of an acid environment EMB forms a complex that precipitates out on the
coliform colonies producing dark centers with metallic sheen.This reaction is
characteristics of E.coli,the major indicator of fecal pollution.
Growth was noted in Brilliant green lactose bile broth both in 44.5c and 37.c
indicating the presence of E.coli. Further confirmation occurred in completes test which
showed Gram negative bacilli. Lactose broth showed acid and gas production.. The
biochemical test showed the presence of E.coli which are Gram negative, flagellated, non
spore forming, Indole positive, Methyl red positive, Voges-Proskaur negative, and Citrate
negative.Menbrane filtration technique also showed a nucleated colonies with metallic
sheen showing the presence of E.coli in EMB plates.
Escherichia coli is a water indicator bacteria and is a sub-group of the coliform
group. Most E.coli are harmless and present in large numbers in the intestines of people
and warm-blooded animals. Some strains, however, may cause illness. The presence
of E.coli in drinking water sample indicates recent faecal contamination indicating the
presence of other pathogens from human fecal contamination. Epidemiological and
microbiological studies have shown that E. coli are better indicator of tropical water
quality (Moe, 2002).
Usually two forms of pollution are taken in to account: toxic chemicals or
pathogenic microorganisms. Probably the largest single source of potentially pathogenic
microbes is animal feces (including human), which contains billions of bacteria per gram.
Although most intestinal microbes are non-pathogenic, some cause serious enteric
disease. The organisms which cause typhoid fever (Salmonella typhi), cholera (Vibrio
cholera), and bacterial dysentery (Shigella flexneri) are examples of enteric diseases
caused by bacteria. In addition, some viral and protozoan pathogens spread through
water contaminated by feces. Water testing for microbiological safety rests on the ability
of microbiologists to detect coliform bacteria.
From the present study it is seen that the most probable number of fecal coliform
is more during the monsoon season followed by post monsoon and it is comparatively
less during the pre-monsoon season. The reason is that during monsoon the flow of water
and the mixing up the domestic sewage with aquatic water body is more and also there is
more mixing up of sediment which is rich in nutrients. Natarajan et al., (1980) also has
observed very low levels of pathogens in estuarine and marine waters during summer
season. Several factors have been proposed which considerably reduce the survival rates
of fecal bacteria in some seasons in aquatic environment. Sunlight is thought to be the
single most important factor contributing to the death of these bacteria in seawater
(Chamberlin, 1978). Other factors include high salinity (Pike,1970),, the presence of
toxic agents (Jones,1964) predation and parasitism (Enzinger,1976) and low nutrition
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6. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012
(Gauthier,1989).In this study the presence Escherichia coli was detected through out the
sampling period which shows that all round the year sewage mixing occurs in
Muttukadu back waters but is more during monsoon season. Sewage accounts for the
greatest volume of waste discharged to estuaries and coastal marine environments (Clark,
1997). More pressing concerns from the human standpoint are an increase in pathogen
density and the associated recreational health risks
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