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A study on the presence of fecal pollution indicator


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A study on the presence of fecal pollution indicator

  1. 1. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012 INTERNATIONAL JOURNAL OF BIOTECHNOLOGY (IJBT)ISSN: (Print)ISSN: (Online) IJBTVolume 1, Issue 1, January- March (2012), pp. 01-07 ©IAEME© IAEME: 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, IndiaABSTRACT Coliform bacteria are a collection of relatively harmless microorganisms that livein large numbers in the intestines of man, warm and cold-blooded animals. A specificsubgroup of this collection is the fecal coliform bacteria, the most common memberbeing Escherichia coli. The presence of fecal coliform bacteria in aquatic environmentsindicates that the water has been contaminated with the fecal material of man or otheranimals. The presence of fecal contamination is an indicator that a potential health riskexists for individuals exposed to this water. The current study is aimed at analyzing thepresence of fecal coliform in Muttukadu back waters, where different kinds of fishes,prawns and shell fishes are harvested for human consumption. The water samples werecollected in pre–monsoon, monsoon and post-monsoon seasons. Water analysis to findthe 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 andbiochemical tests. Analysis showed higher number of fecal indicator in monsoon,followed by post monsoon seasons. The number was comparatively less in pre monsoonseasonINTRODUCTION Water quality is a vital aspect for the survival and well being of the livingresources, especially in the coastal and estuarine areas. Some of these areas are nowunder the direct threat from the increasing load of various pollutants. In order to quantifyand understand the relationship of pollution indicator and human pathogenic bacteriawith environmental factors, several investigators have examined distribution of these1 Department of Micro Biology, Ethiraj College for Women, Egmore, Chennai2 Department of Zoology, Ethiraj College for Women, Egmore, Chennai 1
  2. 2. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012groups of bacteria and certain viruses in coastal waters (Ramaiah and Chandramohan1993 ; Ruiz et al .2000 ; Ramaiah and De 2003 ). Land drainages, domestic sewageoutfalls, and other discharges alter the abundance and type of both autochthonous andallochthonous microbial populations in the near shore environments.Members of twobacteria groups, coliforms and fecal streptococci, are used as indicators of possiblesewage contamination because they are commonly found in human and animal feces.Although they are generally not harmful themselves, they indicate the possible presenceof pathogenic bacteria, viruses, and protozoan that live in human and animal digestivesystems. Therefore, their presence in streams suggests that fecal contamination hasoccurred and other human pathogenic microorganisms might also be present. Swimmingand eating shellfish from the water might be a health risk. Since it is difficult, time-consuming, and expensive to test directly for thepresence of a large variety of pathogens, water is usually tested for total coliforms andfecal coliforms. Faecal coliform bacteria have been widely used as indicators of watercontamination by the fecal material of humans and other warm-blooded animals (APHA1992;Bordalo 1993)Basically water analysis is done for seeing the presence of fecalindicator bacteria which might indicate the presence of other human pathogenic bacterialpopulation of intestinal origin. Marine bivalves accumulate large number of bacteria fromthe 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 themonitoring of sanitary and microbiological quality of water (Gunnison, 1999). TotalColiform and Fecal Coliform counts are the most widely used bacteriological proceduresfor assessment of the quality of drinking and surface waters. Microbiologists rely on theprinciple that higher the incidence of sewage indicator bacteria in any environment,higher would be the chances for human pathogenic bacteria to be present (Brock etal.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 ofIndia and opens into the Bay of Bengal, from where lot of fishes, shellfishes areharvested and the area is surrounded by many aqua cultural farms. Sewage and industrialeffluents from the surrounding area have a greater impact on the Muttukadu back wateraffecting the aquatic animals and in turn the human population. These wastes carryenormous number of microbial pathogens and other heavy metals resulting in greatereconomic loss.MATERIALS AND METHODS Sampling was done during pre-monsoon, monsoon and post monsoon seasons of2010. The water samples were collected in sterile screw capped bottles for water qualityassessment. The water sample was brought to the laboratory in portable icebox with in 2hours. 2
  3. 3. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012MULTIPLE TUBE TEST The presence of faecal coliforms aerogenic Escherichia coli in water isdetermined by means of the multiple tube test procedurePresumptive test Most probable number test was done with three dilutions of the sample. Thedilutions used were 10ml, 1 ml and 0.1ml. Each dilution requires five Macconky brothmedia tubes, thus the sample is inoculated in five replicas of each dilution.. First set of5 tubes with double strength Macconky broth was inoculated with1 0 . 0 m l o f t h e sample to be tested. Second set of another 5 tubes withsingle strength medium with 1.0 ml of the sample and the third set of 5 tubeswith 0.1 ml of the sample using sterile pipettes. After 24-48 hours of incubation at37c ,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 colorindicating the pH change. Small tubes, called Durham’s tubes, were used to collect thegas bubbles formed during the fermentation. The medium in the inverted Durhams tubeswithin the test tubes are replaced by the gas produced, thus enabling the observation. Thevalues were compared with MPN standard chart. The presence of fecal indicatororganism Echerichia Coli is also checked by inoculating in to two tubes of brilliant greenlactose bile broth. One tube was inoculated at 44.5º c and another tube was incubated at37º c for 24 hrs.Confirmed test The presence of fecal indicator Coliform, Escherichia coli is further confirmed bystreaking in Eosin methylene blue agar from the Positive presumptive tube which isfollowed by completed test.Completed test The isolated bacteria from EMB were taken to nutrient agar slant and Lactosebroth with Durham’s tube for acid and gas production .Gram’s staining was done with thegrowth on the nutrient agar slant.MEMBRANE FILTRATION TECHNIQUE One of the easy and fast methods for the analysis of water quality is by usingMembrane filtration technique.100 ml of the sample is passed through a 47 mmmembrane with a pore size of 0.45 µm using a filter funnel and a vacuum system. Any 3
  4. 4. International Journal of Biotechnology (IJBT), Volume 1, Issue 1, January- March 2012organisms in the sample are concentrated on the surface of the membrane. The filter isthen placed in a Petri dish with Eosin methylene blue agar medium.Appearance ofnucleated colony with metallic sheen confirms the presence of Escherichia coli. Furtherbiochemical 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 Sample volume 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 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 4
  5. 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 andmetallic sheen in Eosin methylene blue agar. Eosin methylene blue contains the dyemethylene blue, which inhibits the growth of gram – positive organisms. In thepresence of an acid environment EMB forms a complex that precipitates out on thecoliform colonies producing dark centers with metallic sheen.This reaction ischaracteristics of E.coli,the major indicator of fecal pollution. Growth was noted in Brilliant green lactose bile broth both in 44.5c and 37.cindicating the presence of E.coli. Further confirmation occurred in completes test whichshowed Gram negative bacilli. Lactose broth showed acid and gas production.. Thebiochemical test showed the presence of E.coli which are Gram negative, flagellated, nonspore forming, Indole positive, Methyl red positive, Voges-Proskaur negative, and Citratenegative.Menbrane filtration technique also showed a nucleated colonies with metallicsheen showing the presence of E.coli in EMB plates. Escherichia coli is a water indicator bacteria and is a sub-group of the coliformgroup. Most E.coli are harmless and present in large numbers in the intestines of peopleand warm-blooded animals. Some strains, however, may cause illness. The presenceof E.coli in drinking water sample indicates recent faecal contamination indicating thepresence of other pathogens from human fecal contamination. Epidemiological andmicrobiological studies have shown that E. coli are better indicator of tropical waterquality (Moe, 2002). Usually two forms of pollution are taken in to account: toxic chemicals orpathogenic microorganisms. Probably the largest single source of potentially pathogenicmicrobes is animal feces (including human), which contains billions of bacteria per gram.Although most intestinal microbes are non-pathogenic, some cause serious entericdisease. The organisms which cause typhoid fever (Salmonella typhi), cholera (Vibriocholera), and bacterial dysentery (Shigella flexneri) are examples of enteric diseasescaused by bacteria. In addition, some viral and protozoan pathogens spread throughwater contaminated by feces. Water testing for microbiological safety rests on the abilityof microbiologists to detect coliform bacteria. From the present study it is seen that the most probable number of fecal coliformis more during the monsoon season followed by post monsoon and it is comparativelyless during the pre-monsoon season. The reason is that during monsoon the flow of waterand the mixing up the domestic sewage with aquatic water body is more and also there ismore mixing up of sediment which is rich in nutrients. Natarajan et al., (1980) also hasobserved very low levels of pathogens in estuarine and marine waters during summerseason. Several factors have been proposed which considerably reduce the survival ratesof fecal bacteria in some seasons in aquatic environment. Sunlight is thought to be thesingle most important factor contributing to the death of these bacteria in seawater(Chamberlin, 1978). Other factors include high salinity (Pike,1970),, the presence oftoxic agents (Jones,1964) predation and parasitism (Enzinger,1976) and low nutrition 5
  6. 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 thesampling period which shows that all round the year sewage mixing occurs inMuttukadu back waters but is more during monsoon season. Sewage accounts for thegreatest volume of waste discharged to estuaries and coastal marine environments (Clark,1997). More pressing concerns from the human standpoint are an increase in pathogendensity and the associated recreational health risksREFERENCES 1. APHA (1992) Standard Methods for the Examination of Water and Wastewater, 15th edn. New York: Water Pollution Control Federation 2. Bordalo, A.A. (1993) Effects of salinity on bacterioplankton: field and microcosm experiments. Journal of Applied Bacteriology 75, 393–398 3. Brock,T..,Madigan,M.T.,maetinko,J.M and Parker,J.(1994).Biology of th Microorganism(7 Edition ),Prentice hall ,New Jersey 4. Chamberlin, C. E., and R. Mitchell. 1978. A decay model for enteric bacteria in natural waters, p. 325–348. In R. Mitchell (ed.), Water pollution microbiology, vol. 2. Wiley, New York 5. Clark, R.B. (1997). What is pollution? In R.B. Clark (Ed.), Marine pollution (pp. 1-20). Oxford: Clarendon Press. 6. Enzinger, E. M., and R. C. Cooper. 1976. Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters. Appl. Environ. Microbiol. 31:758–763 7. Fujioka, R.S. (2002). Microbial indicators of marine recreational water quality. In C.J. Hurst, R.L. Crawford, G. R. Knudsen, M.J. McInerney & L.D. Stetzenbach (Eds.), Manual of environmental microbiology (pp. 907-953). Washington DC: ASM Press. 8. Gauthier, M. J., P. M. Munro, and V. A. Breittmayer. 1989. Influence of prior growth conditions on low nutrient response of Escherichia coli in seawater. Can. J. Microbiol. 35:379–383. 9. Gunnison, D., 1999. Evaluating Microbial Pathogens in Reservoirs. Water Quality Technical Notes Collection. US Army Engineer Research and Development Center, USA. 10. Jan A.Olafsen,Helene V,Mikkelsen,Hanne M.Glever,and Gei Hovik Hansen Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures, Applied And Environmental Microbiology, June 1993,p. 1848- 1854 11. Jones, G. E. 1964. Effect of chelating agents on the growth of Escherichia coli in seawater. J. Bacteriol. 87:484–499 12. Moe, C.L. (2002). Waterborne transmission of infectious agents. In C.J. Hurst, R.L. Crawford, G.R. Knudsen, MJ. Mclnerney, & L.D. 6
  7. 7. 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: 97–107. 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, 49–50. 17. Ramaiah, N. and De, J. (2003). Unusual rise in mercury resistant bacteria in coastal environments. Microbial Ecology, 45, 444–454. 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, 49–50. 19. Ramaiah, N. and De, J. (2003). Unusual rise in mercury resistant bacteria in coastal environments. Microbial Ecology, 45, 444–454. 7