International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012

INTERNATIONAL JOURNAL OF BIOTECHNOLOGY (IJBT)

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IJBT
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Volume 1, Issue 1, January- March (2012), pp. 01-07 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 premonsoon, 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 2

Department of Micro Biology, Ethiraj College for Women, Egmore, Chennai Department of Zoology, Ethiraj College for Women, Egmore, Chennai

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 (1247N, 8015E) 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.

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 Durhams 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 44.5 c and another tube was incubated at 37 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 Durhams tube for acid and gas production .Grams 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 m using a filter funnel and a vacuum system. Any 3

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 WATER SAMPLE TABLE: 2 MOST PROBABLE NUMBER WATER SAMPLE

PRE MONSOON SEASON S.no 10 ml + + + + + 543= 275* Sample volume 1 ml + + + + 0.1 ml + + + S.no

MONSOON SEASON Sample volume 10 ml + + + + + 553= 900* 1 ml + + + + + 0.1 ml + + +

1 2 3 4 5 MPN values

1 2 3 4 5 MPN values

Most propable number of Coliforms in 100 ml of water. (+ ) Acid and Gas produced , ( -) No Acid and Gas produced

Most propable number of Coliforms in 100 ml of water. (+ ) Acid and Gas produced , ( -) No Acid and Gas produced

TABLE: 3 MOST PROBABLE NUMBER WATER SAMPLE

A CAMPARISON OF MPN VALUES OF WATER TAKEN IN THREE SEASONS

S.no

POST MONSOON SEASON 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

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 5

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

REFERENCES
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International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012

13. Natarajan, R.M., Abraham and G.B. Nair, 1980. Distribution of Vibrio parahaemolyticus in Porto-Novo environment. India J. Med. Res., 71: 679-687 14. Pike, E. B., A. H. L. Gameson, and D. J. Gould. 1970. Mortality of coliform bacteria in seawater samples in the dark. Rev. Int. Oceanogr. Med. 18/19: 97107. 15. Ramaiah, N. and Chandramohan, D. (1993). Ecological and laboratory studies on the role of luminous bacteria and their luminescence in the coastal pollution surveillance. Marine Pollution Bulletin, 26, 190 - 201 16. Ruiz, G.M., Rawlings, T.K., Dobbs, F.C., Drake, L.A., Mullady, T., Huq, A. and Colwell, R.R. (2000). Global spread of microorganisms by ships. Nature, 408, 4950. 17. Ramaiah, N. and De, J. (2003). Unusual rise in mercury resistant bacteria in coastal environments. Microbial Ecology, 45, 444454. 18. Ruiz, G.M., Rawlings, T.K., Dobbs, F.C., Drake, L.A., Mullady, T., Huq, A. and Colwell, R.R. (2000). Global spread of microorganisms by ships. Nature, 408, 4950. 19. Ramaiah, N. and De, J. (2003). Unusual rise in mercury resistant bacteria in coastal environments. Microbial Ecology, 45, 444454.