Project on Water Potability by Anup Kumar Ojha

PROJECT ON PROJECT ON

r

SOURCES AT SODEPUR, KOLKATA

The Degree of Bachelor of Science in Applied Biotechnology

Under

The Sikkim Manipal University of Health, Medical & Technological Sciences

Project is submitted by

Name: ANUP KUMAR OJHAName: ANUP KUMAR OJHA

Registration Number:05Registration Number:05

Semester: BSc. BT-6thSemester: BSc. BT-6th

Study Centre

ABC FOUNDATIONStudy Center Code 1831

BB – 36, Sector – 1, Salt Lake CityKolkata – 700064

West Bengal

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This is to certify that this project is done under my guidance by Anup Kumar Ojha, a student of Sikkim Manipal University of Health, Medical and Technological Science, Roll No. 05 Bachelor of Science in Applied Biotechnology.

The Project Report has not been submitted for any other examination and does not form a part of other course undergone by the candidate.

Internal Examiner External Examiner

This is to certify that this project is done under my guidance by Anup Kumar Ojha, a student of Sikkim Manipal University of Health, Medical and Technological Science, Roll No. 05 Bachelor of Science in Applied Biotechnology.

The Project Report has not been submitted for any other examination and does not form a part of other course undergone by the candidate.

Internal Examiner External Examiner

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I hereby declare that project report titled

Qualitative Analysis and Determination of MPN Index of various water sample collected from different sources at Sodepur, Kolkata

Submitted to the

SIKKIM MANIPAL UNIVERSITY OF HEALTH, MEDICAL &TECHNOLOGICAL SCIENCES

in partial fulfillment of the requirement of the degree of

Bachelor of Science in Applied Biotechnology

It is my original work and not submitted for the award of any other degree, diploma, or other similar title or prizes.

Signature

KolkataDated: Registration No. 05

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Submitted by:ANUP KUMAR OJHA

VI B.Sc. BiotechnologyRoll No. 05

Subject code: BO0033Centre code: 01831

Sikkim Manipal University

Supervisor:Dr. I. P. Poddar

ChairmanSubhasree Biotech

A Unit of the Calcutta Silk Mfg. Co. Ltd.23, B. T. Road

KOLKATA-700115

Project report submitted for the partial fulfillment of the requirement for qualifying B.Sc. Biotechnology from Sikkim Manipal University

Contents

SL. NO. TOPIC PAGE NO.

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1. Certification of work ---

2. Student deceleration ---

3. Acknowledgement ---

4. Introduction Water quality & Contaminants Sources of drinking water in the Earth Coliform Index Indicator organism Quality of potable water

08 - 12

5. Aim & Objective 13

6. Materials & Method Materials Methods

1. Preparation of culture media2. Test for water potability and

determination of MPN Index3. Gram Staining4. MPN Index

13-18

7. Observation & Result 18 - 22

8. Discussion 23

9. Reference 23 - 24

Acknowledgement

The work embodied in the project has been carried out for partial fulfillment of the requirement for the completion of B.Sc. Biotechnology from Sikkim Manipal University.

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The project has been entitled as “Qualitative Analysis and Determination of MPN Index of Various Water Samples Collected from Different Sources at Sodepur, Kolkata”.

I would like to convey my thanks and regards to Dr. I. P. Poddar, Director of Subhasree Biotech. It is, indeed, for him I learnt a lot during the tenure of my training period.

My special thanks go to Dr. S. Sukul and Dr. P. Datta for providing me this opportunity to work in this highly esteemed laboratory and also for the valued suggestions given from time to time which, I believe, will go a long way with me to build an enshrining future ahead.

It is my extreme pleasure and great opportunity to express my profound sense of gratitude to Mr. Tanmoy Sarkar and Mr. Soummya Seal, Project Associate, Subhasree Biotech for their guidance, constant encouragement, affection and great help in the completion and presentation of this project work.

I also express sincere gratitude to Mr. Sidhartha Banerjee, Mr. Anirban Roy Chowdhury and Mr. Sumit Kumar Dey for their constant inspiration as well as warm and valuable suggestions which helped me a lot to complete this project work.

I also tender my owe to the Director cum Principal Mr. S.K.D Burman, Faculty in-charge Mrs. Sanjogita Basu and other respected faculties of ABC Foundation for granting my long cherished dream to work amidst expertise and state-of-the-art laboratory.

Last but not the least; I thank my parents for their all round support extended at the time of need.

(ANUP KUMAR OJHA)VI B.Sc. Biotechnology Registration No: 520687137Learning Centre Code: 01831ABC FoundationSikkim Manipal University

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1. INTRODUCTION

Water of sufficient quality to be used as drinking water is termed Potable Water. Although many fresh water sources are utilized by humans, some contain disease vectors or pathogens and cause long-term health problems if they do not meet certain water quality guidelines. Water that is not harmful for human beings is sometimes called safe water that is not contaminated to the extent of being unhealthy. The available supply of drinking water is an important criterion for the population supported by the planet Earth.

As of the year 2006 (and pre-existing for at least three decades), there is a substantial shortfall in availability of potable water, primarily arising from overpopulation in lesser developed countries. As of the year 2000, 37% of the populations of lesser developed countries did not have access to safe drinking water [1]. Implications for disease propagation are significant. The World Health Organization (WHO) sets international standards for drinking water.

Typically water supply networks deliver a single quality of water, whether it is to be used for drinking, washing or landscape irrigation; one example is urban China, where drinking water can be optionally delivered by a separate tap. In the United States, public drinking water is governed by the Safe Drinking Water Act (SDWA) that protects the right of employees to report potential violations.

The standard test for bacterial contamination is a laboratory analysis of coliform bacteria, a convenient marker for a class of harmful fecal pathogens. The presence of fecal coliforms (like Escherichia coli) serves as an indication of contamination by sewage.

Over large parts of the world, humans drink water that contains disease vectors or pathogens or contain unacceptable levels of dissolved contaminants or solids in suspension. Such waters are not potable water and drinking such waters or using them in cooking leads to widespread acute, chronic illness and sometimes even death.

1.1. Water quality and contaminants

Throughout most of the world the most common contamination of raw water sources is from human sewage and in particular human faecal pathogens and parasites. In 2006, waterborne diseases were estimated to cause 1.8 million deaths each year while about 1.1 billion people lacked proper drinking water.[1]. It is clear that people in the developing world need to have access to good quality water in sufficient quantity, water purification technology and availability and distribution systems for water. In many parts of the world the only sources of water are from small streams often directly contaminated by sewage. Even wells do not eliminate the risk of contamination.

Most water requires some type of treatment before use. The extent of treatment depends on the source of the water.

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The most reliable way to kill microbial pathogenic agents is to heat water to a boiling [3], but this requires abundant sources of fuel and is very onerous on the households especially where it is difficult to store boiled water in sterile conditions. Other techniques, such as varying forms of filtration, chemical disinfection, and exposure to ultraviolet radiation (including solar UV) have been demonstrated in an array of randomized control trials to significantly reduce levels of water-borne disease among users in low-income countries.

Parameters for drinking water quality typically fall under two categories: chemical/ physical and microbiological. Chemical/ Physical parameters include heavy metals, trace organic compounds, Total Suspended Solids (TSS) and turbidity. Microbiological parameters include Coliform bacteria, E. coli, and specific pathogenic species of bacteria (such as cholera-causing Vibrio cholerae), viruses, and protozoan parasites.

Chemical parameters tend to pose more of a chronic health risk through build-up of heavy metals although some components like nitrates/ nitrites and arsenic may have a more immediate impact. Physical parameters affect the aesthetics and taste of the drinking water and may complicate the removal of microbial pathogens.

Originally, fecal contamination was determined with the presence of coliform bacteria, a convenient marker for a class of harmful fecal pathogens. The presence of faecal coliforms (like E. coli) serves as an indication of contamination by sewage. Additional contaminants include protozoan oocysts such as Cryptosporidium sp., Giardia lamblia, Legionella, and viruses (enteric) [5]. Microbial pathogenic parameters are typically of greatest concern because of their immediate health risk.

1.2. Sources of drinking water in the Earth

Earth's surface consists of 70% water. But sources where drinkable water may be obtained include:

Ground sources such as groundwater, hydrosphere zones and aquifers Precipitation which includes rain, hail, snow, fog, etc. Surface water such as rivers, streams, glaciers Biological sources such as plants The sea through de-salination

As of the year 2000, 27% of the populations of lesser developed countries did not have access to safe drinking water [6]. This proportion has declined steadily over the last decades. Implications for disease propagation are significant.

The lack of water and the lack of hygiene is one of the biggest problems that many poor countries have encountered. The problem has reached such endemic proportions that 2.2 million deaths per annum occur from unsanitary water – 90% of these are children under the age of five [4]. Solar water disinfection is a low-cost method of purifying water that can often be implemented with locally available materials.

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1.3. Coliform Index

The Coliform Index is a rating of the purity of water based on a count of fecal bacteria. Coliform bacteria are microorganisms that primarily originate in the intestines of warm-blooded animals. By testing for coliforms, especially the well known E.Coli, which is a thermo-tolerant coliform, one can determine if the water has probably been exposed to fecal contamination; that is, whether it has come in contact with human or animal feces. It is important to know this because many disease-causing organisms are transferred from human and animal feces to water, from where they can be ingested by people and infect them. Water that has been contaminated by feces usually contains pathogenic bacteria, which can cause disease. Some types of coliforms cause disease, but the coliform index is primarily used to judge if other types of pathogenic bacteria are likely to be present in the water.

The Coliform Index is used because it is difficult to test for pathogenic bacteria directly. There are many different types of disease-causing bacteria, and they are usually present in low numbers which do not always show up in tests. Thermo-tolerant coliforms are present in higher numbers than individual types of pathogenic bacteria and they can be tested for relatively easily.

However, the Coliform Index is far from perfect. Thermo-tolerant coliforms can survive in water on their own, especially in tropical regions, so they do not always indicate fecal contamination. Furthermore, they do not give a good indication of how many pathogenic bacteria are present in the water, and they give no idea at all of whether there are pathogenic viruses or protozoa which also cause diseases and are rarely tested for. Therefore, it does not always give accurate or useful results regarding the purity of water.

1.4. Indicator organism

Indicator organisms are used to measure potential fecal contamination of environmental samples. The presence of coliform bacteria, such as E. coli, in surface water is a common indicator of fecal contamination. Coliform bacteria in water samples may be quantified using the most probable number (MPN) method, a probabilistic test which assumes cultivable bacteria meet certain growth and biochemical criteria. If preliminary tests suggest that coliform bacteria are present at numbers in excess of an established cut-off (the Coliform Index), fecal contamination is suspected and confirmatory assays such as the Eijckman test are conducted.

Coliform bacteria selected as indicators of fecal contamination must not persist in the environment for long periods of time following efflux from the intestine, and their presence must be closely correlated with contamination by other fecal organisms. Indicator organisms need not be pathogenic .

Non-coliform bacteria, such as Streptococcus bovis and Clostridia may also be used as an index of fecal contamination .

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1.5. Quality of potable water

Water quality is the physical, chemical and biological characteristics of water. It is most frequently used by reference to a set of standards against which compliance can be assessed. The most common standards used to assess water quality relate to drinking water, safety of human contact, and for health of ecosystems.

1.5.1. Standards

In the setting of standards, agencies make political and technical/scientific decisions about how the water will be used . In the case of natural water bodies, they also make some reasonable estimate of pristine conditions. Different uses raise different concerns and therefore different standards are considered. Natural water bodies will vary in response to environmental conditions. Environmental scientists work to understand how these systems function which in turn helps to identify the sources and fates of contaminants.

The vast majority of surface water on the planet is neither potable nor toxic. This remains true even if sea water in the oceans (which is too salty to drink) is not counted. Another general perception of water quality is that of a simple property that tells whether water is polluted or not. In fact, water quality is a very complex subject, in part because water is a complex medium intrinsically tied to the ecology of the Earth. Industrial pollution is a major cause of water pollution, as well as runoff from agricultural areas, urban storm water runoff and discharge of treated and untreated sewage (especially in developing countries).

1.5.2. Parameters for measurement

The complexity of water quality as a subject is reflected in the many types of measurements of water quality indicators. Some of the simple measurements are —

Temperature pH

Dissolved Oxygen

Conductivity

Oxygen Reduction Potential (ORP)

More complex measurements that must be made in a lab setting require a water sample to be collected, preserved, and analyzed at another location.

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The following is a list of indicators often measured by situational category:

Physical assessment

Alkalinity Color of water PH Taste and odor (geosmin, 2-methylisoborneol (MIB), etc) Dissolved metals and salts (sodium, chloride, potassium, calcium, manganese,

magnesium) Microorganisms such as fecal coliform bacteria (Escherichia coli),

Cryptosporidium, and Giardia lamblia Dissolved metals and metalloids (lead, mercury, arsenic, etc.) Dissolved organics: colored dissolved organic matter (CDOM), dissolved organic

carbon (DOC) Radon Heavy metals Pharmaceuticals Hormone analogs Temperature Total suspended solids (TSS) Turbidity

Chemical assessment

PH Conductivity (also see salinity) Dissolved Oxygen (DO) Nitrate-N Orthophosphates Chemical oxygen demand (COD) Biochemical oxygen demand (BOD) Pesticides

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2. Aim & Objective

To survey water quality of various samples collected from different sources at Sodepur, Kolkata, West Bengal.

3. Materials & Method

3.1. Materials

3.1.1. Water sample

Water sample was collected from various sources:

Municipal water Canal water

Pond water

Ganga river water

Rain water

Drain water

3.1.2. Culture media preparation

Lactose (SRL), Tryptone (SRL), Yeast Extract (SRL), Bromothymol blue (SRL), Distilled water, Endo agar (Hi-Media), Eosin Methylene blue agar (Hi-Media)

3.1.3. Glass & Plastic wares

Conical flask (BOROSIL & RIVIERA), Petri-plates (RIVIERA & POLY-LAB), Beaker (RIVIERA), Test tube (RIVIERA), Pipette (RIVIERA & POLY-LAB), Measuring Cylinder (POLYLAB & RIVIERA), Durham’s Tube, 1000µl, 100µl and 10µl tips (TARSON), Micro-tip box (TARSON)

3.1.4. Instruments

Autoclave (LAMBDA), Incubator (LAMBDA ORIONIS), Laminar Air Flow (LAMBDA ORIONIS), pH Meter (EI), Hot Air Oven (LAMBDA), Balance (DHONA), 4ºC Freezer (GODREJ), Micropipette (Hi-Media)

3.1.5. Others

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Non-absorbent cotton, Absorbent cotton, Tissue Paper, Marker, Test tube rack, Papers, Rubber band, Spatula, Spirit lamp, Alcohol, Inoculation Loop, Butter Paper, MPN Chart

3.2. Methods

3.2.1. Preparation of culture media

Composition of media:

1. Double Strength Lactose Broth

Lactose- 10g

Tryptone- 10g

Yeast Extract - 5g

Bromothymol Blue- 2ml

Distilled Water- 1000ml

pH- 7.0

2. Single Strength Lactose Broth

Lactose- 5g

Tryptone- 10g

Yeast Extract - 5g

Bromothymol Blue- 2ml

Distilled Water- 1000ml

pH- 7.0

3. Endo Agar

Peptone- 10g

Lactose- 10g

K2HPO4- 3.5g

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Na2SO3- 2.5g

Basic Fuschin- 0.5g

Agar- 20g

Distilled Water- 1000ml

pH- 7.0

4. Eosin Methylene Blue Agar (EMB)

Peptone- 10g

Sucrose- 5g

Lactose- 5g

K2HPO4- 2g

Agar- 2g

Eosin- 4ml

Methylene Blue- 0.06mg

Distilled Water- 1000ml

pH- 7.0

These components were weighed and dissolved in 1000ml of distilled water. After pH set up, they were autoclaved at 121ºC for 15 minutes at 15lb/inch2 pressure prior to its plating on sterile petridishes.

3.2.2. Test for water potability and determination of MPN index

A coliform is facultative and aerobic that ferments lactose to produce gas and is Gram negative, non-sporing rod which can be identified by three different tests.

(a) Presumptive test

(b) Confirmed test

(c) Completed test

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The Presumptive test

In this test, a series of 9 test tubes of lactose broth are inoculated with measured amount of water to see if the water contains any lactose fermenting bacteria that produce gas. If after incubation gas is seen in any of the lactose broth, it is presumed that coliforms are present in the water sample. This test is also to determine the Most Probable Number (MPN) of coliform per 100ml of water.

Confirmed test

In this, test plates of EMB Agar or Endo Agar is inoculated from positive gas tubes. This media inhibits the growth of Gram positive bacteria and helps in distinguishing coliform colonies from non-coliforms. On EMB Agar coliforms produce colonies that give green metallic sheen, while on the Endo Agar they produce pink nucleated colony.

Completed test

This test is to determine whether the isolated form from the agar plate is a coliform or not. The organism is inoculated into a lactose broth and onto a nutrient agar slant. If gas is produced in the lactose tube and a slide from the slant shows the presence of Gram negative, Non-sporing rod; then it is confirmed that coliform is present.

Coliform present may be E. coli or Enterobacter aerogens; of the two E. coli is the better sewage indicator since E. aerogens can be of non-sewage origin. To differentiate between these two species IMViC tests are performed.

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3.2.3. Gram Staining

A slide is de-greased and a loopful of culture is smeared on it

Air dry, add Crystal Violet and retain for 60 sec. Wash

Add Gram’s iodine, keep for 60 sec.

Rinse with Gram’s decolorizer (95% Ethanol)

Add Safranin and keep for 30-60 sec. Wash

Air dry and observe under 10X, 45X and 100X respectively

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3.2.4. MPN index

Number of tubes giving positive reaction out of

MPN index per 100ml

95% confidence limits

3 of 10ml each

3 of 1ml each

3 of 0.1ml each

Lower Upper

0 0 1 3 <0.5 9

0 1 0 3 <0.5 13

1 0 0 4 <0.5 20

1 0 1 7 1 21

1 1 0 7 1 23

1 1 1 11 3 36

1 2 0 11 3 36

2 0 0 9 1 36

2 0 1 14 3 37

2 1 0 15 3 44

2 1 1 20 7 89

2 2 0 21 4 47

2 2 1 28 10 150

3 0 0 23 4 120

3 0 1 39 7 130

3 0 2 64 15 380

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3 1 0 43 7 210

3 1 1 75 14 230

3 1 2 120 30 380

3 2 0 93 15 380

3 2 1 150 30 440

3 2 2 210 35 470

3 3 0 240 36 1300

3 3 1 460 71 2400

3 3 2 1100 150 4800

4. Results and Observation

To determine water quality and to calculate its MPN index, samples were inoculated in 3 DSLB tubes containing 10ml sample each, 3 SSLB tubes containing 1ml sample each and 3 SSLB tubes containing 0.1ml sample each. Lactose Broth contains an indicator Bromothymol Blue that changes its color to blue (Alkaline) to yellow (Acidic) as pH of the medium declines. Durham’s tube is used to trap any gas bubbles formed.

Coliforms are known as ‘Mixed acid fermenters’. They ferment lactose to acid and gas. Acid changes the color of the lactose broth and gas is trapped in the Durham’s tube. Hence, from visualizing the changed color of the media and trapped gas it is assumed that the sample may be contaminated with sewage.

Sample-1: Municipal Water

Color of the media in all tubes remained blue and no gas bubble was seen in the Durham’s tube.

Sample-2: Canal Water

Color of the media in all tubes changed from blue to yellow and gas bubble was seen in the Durham’s tube.

Sample-3: Pond Water

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Color of the media in all tubes changed from blue to yellow and gas bubble was seen in the Durham’s tube.

Sample-4: Ganga River Water

Color of the media in 2 DSLB tubes and 1 SSLB tube (containing 1ml of the sample) changed from blue to yellow and the remaining test tubes retained the blue color of the media. Gas bubble was seen in the Durham’s tubes of those tubes in which color of the media changed from blue to yellow.

Sample-5: Rain Water

Color of the media in 2 DSLB tubes, 2 SSLB tubes (containing 1ml of the sample) and 1 SSLB tube (containing 0.1ml of the sample) changed from blue to yellow and the remaining test tubes retained the blue color of the media. Gas bubble was seen in the Durham’s tubes of those tubes in which color of the media changed from blue to yellow.

Sample-6: Drain Water

Color of the media in all tubes changed from blue to yellow and gas bubble was seen in the Durham’s tube.

The over all observations are summed here in the following table:

Sample

No. of tubes showing positive resultMPN

index per 100ml

Lower limit

Upper limit

DSLB (10ml)

SSLB (1ml)

SSLB (0.1ml)

Municipal Water

1 0 0

4 <0.5 20

Canal Water

3 3 3

1100 150 4800

20

Pond Water

3 3 3

1100 150 4800

Ganga River Water

2 1 0

15 3 44

Rain Water

2 2 1

28 10 150

Drain Water

3 3 3

1100 150 4800

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Figure 1: DSLB and SSLB possess Bromothymol blue as an internal indicator that changes its color as pH of the medium declines from alkaline to acidic range; also Durham’s tube is inserted to trap gas bubbles produced, if any. In the negative tube [Fig-1(a)], lactose was not fermented to acid and gas, hence color of the media remained blue (the color of the indicator in an alkaline solution) and no gas was trapped in the Durham’s tube. On the contrary, in the positive tube [Fig- 1(b)], lactose was fermented to acid (there by changing the color of the medium to yellow from blue) and gas (trapped in the Durham’s tube).

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Figure 2: Determination of MPN requires samples to be inoculated in 3 DSLB tubes containing 10ml sample each, 3 SSLB tubes containing 1ml sample each and 3 SSLB tubes containing 0.1ml sample each. Fig-2 shows two complete set ups. Fig-1(a) shows negative result as color of the medium remained blue and no gas bubbles were produced, while Fig-1(b) shows a positive result as color of the medium changed from blue to yellowish with decline in pH and gas bubbles were trapped in the Durham’s tubes.

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5. Discussion

During the course of studying water quality of various samples by determining MPN index, we got a mixed result.

Municipal water sample has a very low MPN index (4) and hence is not contaminated with sewage. Therefore its potability is unquestionable.

Ganga water has an MPN index of 15. This indicates that Ganga water is sewage polluted. But since the load is very less, the water can be decontaminated by commercially available water purification process to convert it into human consumable water.

Rain water has an MPN index of 28. This can be used as a potential substitute to potable water. Natural water bodies are bearing the brunt of high pollution level. Also ground water level is declining. In this juncture if rain water can be stored and decontaminated, it can meet the massive demand to supply safe potable water.

Canal water, pond water and drain water in the surveyed locality have the highest MPN index (1100) and hence they are highly contaminated with sewage and therefore not fit to drink at all.

6. References

1. U.S. Centers for Disease Control and Prevention. Atlanta, GA (March 2006). “Safe Water System: A Low-Cost Technology for Safe Drinking Water.” Fact Sheet, World Water Forum 4 Update.

2. Centre for Affordable Water and Sanitation Technology (March 2008), “Household Water Treatment Guide,”

3. WHO’s Guidelines for Drinking Water Quality 4. Clasen, T., Schmidt, W., Rabie, T., Roberts, I., Cairncross, S (Published 12 March

2007). Interventions to improve water quality for preventing diarrhea: a systematic review and meta-analysis. British Medical Journal, doi:10.1136/bmj.39118.489931.BE

5. U.S. EPA. “Drinking Water Contaminants: Microorganisms.” 6. I.A. Shiklomanov (2000), Appraisal and Assessment of World Water Resources,

Water International 25(1): 11-327. Conroy R.M., Meegan M.E., Joyce T., McGuigan K., Barnes J. (1999), solar

disinfection of water reduces diarrhoeal disease, an update, Arch Dis Child, Vol. 81.

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8. Conroy R.M., Meegan M.E., Joyce T.M., McGuigan K.G., Barnes J. (2001) Use of solar disinfection protects children under 6 years from cholera. Arch Dis Child; 85:293-295

9. Rose A. at al. (2006). Solar disinfection of water for diarrhoeal prevention in Southern India. Arch Dis Child, 91(2): 139-141

10. Hobbins M. (2003). The SODIS Health Impact Study, Ph.D. Thesis, Swiss Tropical Institute Basel

11. United Nations Children’s Fund (UNICEF, Sep, 2006). New York, NY. “Safe Drinking Water.” Excerpt from “Progress since the World Summit for Children: A Statistical Review.”

12. March 2008, Cashing in on Climate Change, IBIS World 13. United States Environmental Protection Agency (EPA, 2006). Washington, DC.

“Water Quality Standards Review and Revision.” 14. Oblinger, J. L. and J. A. Koburger, J. A. (1975). “Understanding and Teaching the

Most Probable Number Technique”. J. Milk Food Technol. 38(9), 540-545.

________________

THANK YOU

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