MICROBIAL MICROBIAL ASSESSMENT OF ASSESSMENT OF

MADIVALAMADIVALA AAMADIVALA MADIVALA –– A A REJUVENATED LAKE IN REJUVENATED LAKE IN

BANGALORE URBAN BANGALORE URBAN DISTRICTDISTRICTBy Dr.N.Nandini,By Dr.N.Nandini,

AnupamaB.SAnupamaB.SppAboud S. JumbeAboud S. Jumbe

Madivala lakeMadivala lakeMadivala lake Madivala lake

IntroductionIntroduction

Wetlands are important ecosystems and are diverse inWetlands are important ecosystems and are diverse inWetlands are important ecosystems and are diverse in Wetlands are important ecosystems and are diverse in terms of habitats, biota, distribution, functions, and terms of habitats, biota, distribution, functions, and usesuses

Tanks in and around urban areas of Bangalore Tanks in and around urban areas of Bangalore receive considerable amount of sewage inflow. Thisreceive considerable amount of sewage inflow. Thisreceive considerable amount of sewage inflow. This receive considerable amount of sewage inflow. This has caused a severe and persistent microbial pollution has caused a severe and persistent microbial pollution in the urban tanks of the city. But as the urban in the urban tanks of the city. But as the urban sprawling continues the number of live tanks insprawling continues the number of live tanks insprawling continues, the number of live tanks in sprawling continues, the number of live tanks in Bangalore has also dwindled from 262 in the middle of Bangalore has also dwindled from 262 in the middle of last century to just about eightylast century to just about eighty--one tank. (Kumar one tank. (Kumar 2000)2000)2000), 2000),

Cont.,Cont.,

This combination of several environmental pressures This combination of several environmental pressures poses a serious threat to the ecological survival of poses a serious threat to the ecological survival of th l k ll th h lth f b d llth l k ll th h lth f b d llthese lakes as well as the health of urban dwellers these lakes as well as the health of urban dwellers living around these contaminated systems. living around these contaminated systems. Regular monitoring of water bodies with requiredRegular monitoring of water bodies with requiredRegular monitoring of water bodies with required Regular monitoring of water bodies with required number of environmental parameters including that of number of environmental parameters including that of bacteriological characteristics can help prevent bacteriological characteristics can help prevent outbreak of diseases and occurrence of health outbreak of diseases and occurrence of health hazards associated to aquatic pollution. hazards associated to aquatic pollution. Hence water quality survey and monitoring programsHence water quality survey and monitoring programsHence water quality survey and monitoring programs Hence water quality survey and monitoring programs are essential to estimate the pollution level at which are essential to estimate the pollution level at which additional pollutants are getting added and causing additional pollutants are getting added and causing p g g gp g g gpollutionpollution..

ColiformsColiformsColiformsColiforms

Coliforms include all aerobic and facultativeColiforms include all aerobic and facultativeColiforms include all aerobic and facultative Coliforms include all aerobic and facultative anaerobic,Ganaerobic,G--ve nonsporeforming bacilli that ve nonsporeforming bacilli that when incubated at 35when incubated at 3500c, can ferment lactose c, can ferment lactose and produce gas within 48hrs.Coliform group and produce gas within 48hrs.Coliform group contains members of contains members of Escherichia,Escherichia,CitrobacterCitrobacterKLebsiellaKLebsiellaSerratia and EnterobacterSerratia and EnterobacterSerratia and EnterobacterSerratia and Enterobacter

objectiveobjectiveobjectiveobjective

The main objective of this study was to measureThe main objective of this study was to measureThe main objective of this study was to measure The main objective of this study was to measure the level of faecal contamination in Madiwala the level of faecal contamination in Madiwala Lake using MPN and MF techniquesLake using MPN and MF techniquesTo isolate, identify and confirm the presence of To isolate, identify and confirm the presence of E.coli , Enterococci and Enterobacter aerogenes E.coli , Enterococci and Enterobacter aerogenes in Lake water samplesin Lake water samples. .

MATERIALS AND METHODSMATERIALS AND METHODSMATERIALS AND METHODSMATERIALS AND METHODS

Study AreaStudy Area::Study AreaStudy Area::Madiwala Lake is located in the southern fringes Madiwala Lake is located in the southern fringes of the Bangalore city It is one of the biggestof the Bangalore city It is one of the biggestof the Bangalore city. It is one of the biggest of the Bangalore city. It is one of the biggest lakes in the city and is situated bewteen 12lakes in the city and is situated bewteen 12°° 54' 54' 28" North, 7728" North, 77°° 37' 0" East in Bangalore city.37' 0" East in Bangalore city.g yg y. The lake covers full extension of 114.16 Ha . The lake covers full extension of 114.16 Ha and a water spread area of 780,802 mand a water spread area of 780,802 m22

It is a home to many migratory birdsIt is a home to many migratory birdsLike spot billed pelican & egrets.Like spot billed pelican & egrets.p p gp p g

Sample Collection:Sample Collection:

Water samples from four fixed stations of Water samples from four fixed stations of ppMadiwala Lake were collected in the month of Madiwala Lake were collected in the month of September 2007 when the monsoon season September 2007 when the monsoon season was at its peakwas at its peakwas at its peak was at its peak The samples were collected in suitable bottles The samples were collected in suitable bottles that had been previously cleaned and rinsed inthat had been previously cleaned and rinsed inthat had been previously cleaned and rinsed in that had been previously cleaned and rinsed in distilled water and sterilized by autoclaving distilled water and sterilized by autoclaving before mounted for sampling. before mounted for sampling. All the samples were stored in cool ice box and All the samples were stored in cool ice box and immediately brought to the laboratory within one immediately brought to the laboratory within one hour ready for analysishour ready for analysishour ready for analysis.hour ready for analysis.

Membrane Filter Technique (MFTMembrane Filter Technique (MFT))::

The water sample was filtered through the The water sample was filtered through the p gp gcellulose filters having the porosity of 0.45 cellulose filters having the porosity of 0.45 µµm m and diameter of 47 mm.and diameter of 47 mm.A b fil h l d MA b fil h l d M E dE dA membrane filter was then placed on MA membrane filter was then placed on M-- Endo Endo Agar plate (recommended for detection of Agar plate (recommended for detection of coliform and other enteric organisms) andcoliform and other enteric organisms) andcoliform and other enteric organisms) and coliform and other enteric organisms) and incubated at 35incubated at 3500C and 45C and 4500C for 24 C for 24 –– 48 hours48 hoursThe presence of Enterococci in the lake sample The presence of Enterococci in the lake sample was also confirmed by using Enterococcus was also confirmed by using Enterococcus confirmatory agar.confirmatory agar.

Most Probable Number (MPN) TechniqueMost Probable Number (MPN) Technique::Most Probable Number (MPN) TechniqueMost Probable Number (MPN) Technique::

The standard test to estimate the number of coliform The standard test to estimate the number of coliform groups may be carried out by multiple tube dilution groups may be carried out by multiple tube dilution technique technique There are three steps to estimate the coliform groups There are three steps to estimate the coliform groups by MPN technique by MPN technique ––Presumptive TestPresumptive TestConfirmatory TestConfirmatory TestCompleted Test Completed Test

Cont.,Cont.,Cont.,Cont.,

For a For a Presumptive TestPresumptive Test, single strength and , single strength and pp , g g, g gdouble strength lauryl tryptose broth were double strength lauryl tryptose broth were prepared, sterilized in a test tube containing prepared, sterilized in a test tube containing Durham’s tubes to show the gas productionDurham’s tubes to show the gas productionDurham s tubes to show the gas production. Durham s tubes to show the gas production. Finally all the tubes were incubated at 37Finally all the tubes were incubated at 3700C for a C for a period of 24period of 24 –– 48 hours The tubes were48 hours The tubes wereperiod of 24 period of 24 48 hours. The tubes were 48 hours. The tubes were analyzed after the incubation time for gas analyzed after the incubation time for gas production and acid production along with production and acid production along with t biditt biditturbidity.turbidity.

Contd……Contd……

In the In the confirmatory testconfirmatory test, the water samples from , the water samples from all the presumptive tests with lauryl tryptoseall the presumptive tests with lauryl tryptoseall the presumptive tests with lauryl tryptose all the presumptive tests with lauryl tryptose broth tubes were inoculated into tubes of brilliant broth tubes were inoculated into tubes of brilliant green lactose bile broth (BGLB) with Durham’sgreen lactose bile broth (BGLB) with Durham’sgreen lactose bile broth (BGLB) with Durham s green lactose bile broth (BGLB) with Durham s tubes and incubated at 35tubes and incubated at 3500C for 48 hours. After C for 48 hours. After incubation the formation of gas signifies the incubation the formation of gas signifies the positive confirmed test.positive confirmed test.

Cont.,Cont.,Cont.,Cont.,

In theIn the completed testcompleted test, the samples from the, the samples from theIn the In the completed testcompleted test, the samples from the , the samples from the positive BGLB broth from the confirmatory tests positive BGLB broth from the confirmatory tests were streaked onto selective differential medium were streaked onto selective differential medium (EMB) agar for coliforms and inoculated in (EMB) agar for coliforms and inoculated in lactose broth tube lactose broth tube –– and also streaked onto the and also streaked onto the

ffnutrient agar plate to perform gram stainingnutrient agar plate to perform gram staining. .

RESULTS AND DISCUSSIONRESULTS AND DISCUSSIONMembrane Filter TechniqueMembrane Filter Technique

InIn thisthis method,method, itit waswas observedobserved thatthat thethe goldengolden,, gggreengreen coloniescolonies ofof coliformscoliforms withwith metallicmetallic sheensheenshowedshowed aa luxuriantluxuriant growthgrowth atat 353500CC onon MM--EndoEndo AgarAgar(M(M11061106)) atat thethe incubationincubation timetime ofof 2424 –– 4848 hourshours..

EnterobacterEnterobacter aerogenesaerogenes waswas alsoalso observedobserved onon MM--E dE d AA PlPl i hi h i ki k dd idid l il iEndoEndo AgarAgar PlatePlate withwith pinkpink andand mucoidmucoid coloniescoloniesThe presence of Enterococcus faecalis was also The presence of Enterococcus faecalis was also

b d h i ll l l i ith l i tb d h i ll l l i ith l i tobserved showing yellow color colonies with luxuriant observed showing yellow color colonies with luxuriant growth.growth.

Most Probable Number TestMost Probable Number TestMost Probable Number TestMost Probable Number Test

TheThe presumptive testpresumptive test results of differentresults of differentThe The presumptive testpresumptive test results of different results of different volumes of the lake samples after the volumes of the lake samples after the incubation at 35incubation at 3500C for C for 24 to 48 hours using 24 to 48 hours using gglauryl tryptose broth indicated significant lauryl tryptose broth indicated significant color change, gas production, and turbidity color change, gas production, and turbidity Confirmed resultsConfirmed results showed confirmed gas showed confirmed gas production in brilliant green lactose bile production in brilliant green lactose bile b th i L t Bil b th t 35b th i L t Bil b th t 3500broth using Lactose Bile broth at 35broth using Lactose Bile broth at 3500CCfor48 hours for48 hours

Cont.,Cont.,Cont.,Cont.,

TheThe completed Resultcompleted Result with EMB Agarwith EMB AgarThe The completed Resultcompleted Result with EMB Agar with EMB Agar Inoculated with positively confirmed test Inoculated with positively confirmed test tube incubated for 24 hours at 35tube incubated for 24 hours at 3500CCtube incubated for 24 hours at 35tube incubated for 24 hours at 35 CCshowed Greenish Metallic Sheen Colonies showed Greenish Metallic Sheen Colonies while the Coliform colonies inoculated onwhile the Coliform colonies inoculated onwhile the Coliform colonies inoculated on while the Coliform colonies inoculated on Nutrient Agar Nutrient Agar –– slant and a broth tube slant and a broth tube incubated for 48 hours at 35incubated for 48 hours at 3500CC showedshowedincubated for 48 hours at 35incubated for 48 hours at 35 CC showed showed growth as observed on slant with gas growth as observed on slant with gas production in the broth tubeproduction in the broth tubeproduction in the broth tube production in the broth tube

Gram staining results showedGram staining results showed Gram negativeGram negativeGram staining results showed Gram staining results showed Gram negative Gram negative non sporing rodsnon sporing rods in the tested samples in the tested samples There were 264 CFU/100 ml for E.coli in the lake There were 264 CFU/100 ml for E.coli in the lake sample,198CFU/100 ml for Enterobacter sample,198CFU/100 ml for Enterobacter aerogenes and 78 CFU/100 ml for Enterococci aerogenes and 78 CFU/100 ml for Enterococci For recreational fresh waters, Water quality For recreational fresh waters, Water quality guidelines for 33 Enterococci is 33 guidelines for 33 Enterococci is 33 CFU/100 l d f E li i i 126 CFUCFU/100 l d f E li i i 126 CFUCFU/100ml,and for E.coli it is 126 CFU CFU/100ml,and for E.coli it is 126 CFU /100ml(APHA 2005)./100ml(APHA 2005).

Table 2. Membrane Filtration Technique using M - Endo Agar (M1106)(M1106)

I b i

Organisms Growth Colonies

Incubation Time Temperature

Organisms Growth Colonies

35oC 450C

Goldengreencolonies

Coliforms Luxuriantwithmetallicsheen

24 – 48 hours. ++ +

Note: ++ = Higher Growth; + = Less Growth

Table 3. MFT Confirmatory Test Using Enterococcus Confirmatory Agar (M392)

Incubation Time

Organisms Growth Colonies

Temperature

35oC 450CEnterococcusf li

Luxuriant

YellowColor

24 – 48hours. ++ +

fecalis

Note: ++ = Higher Growth; + = Less Growth

Table 4. MPN Test: Presumptive Test Results After 24 and 48 Hours of Incubation

Lake Sample Color Change

GasProduction Turbidityg

24 hours

48 hours

24 hours

48 hours

24 hours

48 hourshours hours hours hours hours hours

Single Strength Medium for 0 1 ml + + + + + +Medium for 0.1 ml

Single Strength Medium for 1ml + + + + + +Medium for 1ml

Double Strength Medium for 10 ml + + + + + +

++ = Higher Growth;+ = Less Growth

Table 6. MPN Test: Completed Test Results After 24 and 48 Hours of Incubation

Agar used

Incubation and

Temperatu ResultsTemperature

EMB Agar Inoculated withpositively confirmed test

Incubationat 24 hours Greenish Metallic

Sheen Coloniestube at 35oC Sheen Colonies

Coliform colonies Incubation Growth as observed onColiform coloniesinoculated on NutrientAgar – slant and a broth

Incubationat 48 hoursat 35oC

Growth as observed onslant with gasproduction in the broth

tube tube.

conclusionconclusionconclusionconclusion

From the above test observations, it is generallyFrom the above test observations, it is generallyFrom the above test observations, it is generally From the above test observations, it is generally concluded here that environmental status of concluded here that environmental status of Madiwala Lake with respect to microbial Madiwala Lake with respect to microbial pollution is continuing to deteriorate. pollution is continuing to deteriorate. The presence of lethal pathogens such as E.coli The presence of lethal pathogens such as E.coli in the lake at dangerous and epidemic levels in the lake at dangerous and epidemic levels underpins the failure of the restoration efforts underpins the failure of the restoration efforts and especially the ineffectual operations of theand especially the ineffectual operations of theand especially the ineffectual operations of the and especially the ineffectual operations of the nearby waste water treatment plant located in nearby waste water treatment plant located in the southern part of the lakethe southern part of the lakethe southern part of the lake. the southern part of the lake.

Cont.,Cont.,Cont.,Cont.,

Moreover, lake encroachment continuesMoreover, lake encroachment continuesMoreover, lake encroachment continues Moreover, lake encroachment continues unabated with raw sewage spilling openly unabated with raw sewage spilling openly across the western buffer zone of the lake into across the western buffer zone of the lake into the water. the water.

Thank youThank youThank youThank you