A PROJECT REPORT

ON

DISTRICT WATER RESOURCES QUALITY STATUS

HELD AT

St. COLUMBAS COLLEGE

HAZARIBAG From NovemberJanuary Session (2008-09)
SUBMITTED BY: ROLL.NO: VOCB.Sc PART III BIOTECN OLOGY DEPARTMENT St . COLUMBAS COLLEGE HAZARIBAG

CERTIFICATE
This to certify that _________a student of B.Sc Part-III, BIOTECHNOLOGY DEPARTMENT (VOCATIONAL), Roll no: voc- _______ has completed her/his project work held at St. Columbas College, Department of Biotechnology, Hazaribag, from 8th December to 30th March . He/She has presented a report of the job training and is here by approved as a credible study carried out, presented and fulfilled for the prescribed syllabus of Vinoba Bhave University, Hazaribag for Biotechnology honors. I as coordinator certify that he/she has successfully performed his/her assigned project.

(Dr. M. A. Mallick) Coor dinator Biotechnology Department St. Columbas College Hazaribag

ACKNOWLEDGEMENT
I, express my indebted gratitude and thanks to the principal of St. Columbas College, Hazaribag
2

 for his support and guidance extended To me during my short-term project work from of December. To 30th March.

8th

I would like to thanks Dr. Anwar Mallick, Coordinator of Biotechnology, St. Columbas College, Hazaribag, for providing all the arrangements necessary for the completion of project and for his support throughout the programmers. I also extend my gratitude to Gautam sir and all other teachers for their valuable and constant involvement in project works.

INDEX
INTRODUCTION: SAMPLE COLLECTION: PHYSICAL TESTS: pH test. CHEMICAL TESTS: Chloride test. Phosphorus test. Residual Chlorine test. Ammonia test. Hardness test. BIOLOGICAL TESTS: Dissolved oxygen (DO) test. Most Probable Number (MPN). a) Presumptive test. b)Confirmed test. c) Completed test. CONCLUSION:

Introduction:
ORIGIN OF THE RESEARCH PROBLEM In Hazaribag town water borne disease are very common, people of the town are being infected on the regular basis, and sometimes to death also. This is an indication that the water that is being used is contaminated with pathogenic microbes. These data led to the development of the idea to work on a project, which will analyze the water quality from different sources i.e. Well, boring supply.

METHODOLOGY:

1] Collection of samples. Precautions. Source. Area.

2] Quality of water will be analyzed under the following heading.

Biological- Coliforms / Pathogens. Physical- pH. Chemical- Chlorine, Hardness, Phosphorus, Residual

chlorine, Ammonia.

SAMPLE COLLECTION
The sample to be analyzed was collected from four different sources that are mainly used for domestic purposes.
Well water. Boring water. Supply water. Distilled water.

Area
Sample was collected from different areas of to get a complete picture of the potability of the water used in Hazaribag town. WATER FROM FOLLOWING AREAS WAS SAMPLED:
1. Ramnagar 2. Suresh colony 3. Cemetery road 4. Haranganj 5. Babugaun 6. Pugmil 7. Matwari 8. Barabazar 9. Aryanagar 10. Anandpuri

Precautions
The vessel used for sample collection should be sterilized.

The source from where sample is collected should be cleaned first. Before collecting the water, let some water runoff from the source.

CHEMICAL TESTS

EXPERIMENT NO.- 1
Aim: To determine the amount of chloride present. Significance:
Chloride, in the form of chloride ion (Cl-), is one of the major inorganic anion in natural water and wastewater.in potable water, the salty taste produced by concentrations is variable and dependent on the chemical composition of water. The chloride concentration is higher in domestic sewage than raw water because sodium chloride (Na Cl) is common item of diet and passes unchanged through the digestive system.

Sources:
Discharges of effluents from chemical industries, sewage discharge, irrigation drainage, contamination from refuse leachates and sea water intrusion in coastal areas are some vital source of excess chloride levels.

Effects:
A high chloride content has deleterious effects on metallic pipes and structures as well as on agricultural crops.It can also corrode concrete by extracting calcium in the form of calcide.

Principle:
Chloride forms white precipitate of silver chloride in a natural or slightly alkaline solution upon titration with standard silver nitrate.Potassium chromate is used as an indicator

Requirements:
Apparatus required Test tube Micropipettes

Sample collection bottle pH paper

Chemicals required Potassium chromate Molecular weight = 169.87 Silver nitrate Molecular weight = 194.20 For 1 M solution -----10 ml AgNo3 = 1.69gm 10ml K2CrO4 = 1.94gm

Procedure:
Transfer 5ml of water sample in a test tube. Check Ph of the solution, which should be between 7 and 10.Add HCl or ammonia to adjust pH. Add 1 drop of potassium chomate to the sample. Take 1 ml of silver nitrate solution in 1ml micropipette and recap the bottle. Titrate until permanent brick red color appears.Note the amount of silver nitrate consumed to calculate the value of chloride in water. Compare whether sample is more turbid or less turbid than 10 NTU.

Permissible limits Drinking water = 250 to 1000 mg/l.

RESULTS:
The amount of chlorine in water samples were found within permissible limits.

SOURCE(mg/l) AREA
Ramnagar Suresh colony Cemetery road Haranganj Babugaun Pugmil Matwari Barabazar Aryanagar Anandpuri WELL 373 435 564 509 543 443 564 667 543 509 SUPPLY 806 760 733 750 678 609 732 690 733 760 BORING 478 455 500 520 570 490 550 706 499 550

Precautions:

Do not touch the silver nitrate. In case of any accidental spills on the body, wash off immediately with water.

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EXPERIMENT NO. -2
Aim: To determine the amount of phosphorus in water. Significance:
Phosphorous in the elemental form is particularly toxic and is subject to bioaccumulation in much the same way as mercury. Phosphorous as phosphate is an essential element for life as a nutrient (for plant growth) and as a key element in the metabolic processes of all living organisms. In natural low concentrations, phosphorous is a limiting factor regulating the growth of algae and plants in water bodies.

Sources:
Natural rocks, domestic sewage, tree leaves, human and animal wastes, agriculture drainage farm and lawn fertilizers, distribution of wetlands, certain industrial wastewater and detergents.

Effects:
In excess of a critical concentration,phosphate stimulates or promotes nuisance algae and plant growth.This algal growth imparts undesirable tastes and odours to water,interferes with water treatment and becomes aesthetically unpleasant.Such a situation is also associated with a condition of accelerated eutrophication or ageing of water.Phosphorous as is not harmful to the organisms.

Principle:

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Phosphorous when reacts with Ammonium molybdate form molybdophosphoric acid and is reduced by stannous chloride to intensely coloured molybdenum blue.

Requirements:
Apparatus required Micropipettes Test tube Sample collection bottle Chemicals required Ammonium molybdate Stannous chloride Hcl Alcohol

Procedure:
Wash the test tube with 2ml Dil.HCl and then wash thoroughly with distilled water. Transfer 12ml of water sample. Take 0.4ml of ammonium mloybdate using 2ml micropipettes and shake well. Add 2 drops of stannous chloride and shake it again. Wait for 10 mins. For color development. Permissible limits River water = 0.1 mg/l Marine or estuarine = 0.1 mg/l

Observations:
SOURCE AREA Ramnagar Suresh colony Cemetery road WELL normal normal normal SUPPLY normal normal normal BORING normal normal normal

12

Haranganj Babugaon Pugmil Matwari Barabazar Aryanagar Anandpuri Distilled water

normal normal normal normal normal normal normal normal

normal normal normal normal normal normal normal normal

normal normal normal normal normal normal normal normal

Result:
The amount of phosphorus in water samples were found within permissible limits.

Precautions:
The glassware used for this test should be properly washed and cleaned. Avoid washing glassware with soap or detergent as phosphorous present in the detergent gets absorbed on the glass surface.

13

EXPERIMENT NO. -3
AIM: To determine the amount of residual chlorine in water. Significance:
Chlorine is widely used for disinfection of water, for deodorization and is cheaply available.For effective disinfection, dose of chlorine, optimum contact period and residual chlorine is required to be known.

Sources:
Chlorine is not a natural constituent of water. Free available chlorine (HOCl and OCl) and combined chlorine (mono- and dichloroamines) appear transiently in surface water or ground water as a result of disinfection of domestic sewage or from industrial processes that use chlorine bleaching operations or to control organisms that grow in cooling water systems.

Effects:
Chlorine combines with water to form hypochlorous and hyochloric acid.Hypochlorous acid dissociates to give OCl- ions.Quantities of OCl- and HOCl- depend on pH of the solution.Hypochlorites also gives OCl- ions.HOCl- rupture the cell membrane of microbes,the diseases producing organism. The presence of excess chlorine intensifies the taste and odour of many other compounds such as phenols etc.It may be harmful to many aquatic organisms in combination with ammonia. 14

Principal:
O-toludine forms yellow complex, the intensity of the developed colour is dependent on the amount of residual chlorine present in chlorine present in the water sample.

Requirements:
Test tubes Sample collection bottle O-toludine pH paper

Procedure:
Transfer 10ml of water sample in a test tube. Check Ph of the sample. If less than 9, add 4 drops of o-toludine if ph is greater than 9 add 8 to10 drops. Compare the colour with colour chart

Observation:
AREA Ramnagar Suresh colony Cemetery road Haranganj Babugaon Pugmil Matwari Barabazar Anandpuri Aryanagar Distilled water WELL normal normal normal normal normal normal normal normal normal normal normal SOURCE SUPPLY normal normal normal normal normal normal normal normal normal normal normal BORING normal normal normal normal normal normal normal normal normal normal normal

Result:
15

Residual chlorine was found absent in the water sampled.

Precautions:
The use hand of hand gloves is recommended. O-toludine is a hazardous chemical. Handle it carefully. Avoid its contact with skin, eyes or any other sensitive body parts.

EXPERIMENT NO. -4
AIM: To determine the amount of ammonia in water.
Significance:
Ammonia comes to groundwater and surface water mainly through human activities and natural decay process. Its concentration is generally low in ground water because it absorbs to soil particles and clays and not leached readily from soil. Presence of ammonia signifies organic pollution. Presence of ammonia also indicates a recent origin of sewage pollution. As the times lapse, the ammonia oxidizes to other forms and will be represented by the presence nitrite and nitrate forms of the pollutant.

Sources:
The most important sources of the presence of ammonia is ammonification of organic matter. Possible sources: sewage discharges, industrial discharges, decay of plant and animal litter, runoff from agricultural fields containing organic fertilizers,etc.

Effects:
Ammonia in higher concentrations is harmful to fish and other biota and even human beings. The toxicity of ammonia increases pH: at higher pH,most of the ammonia remains in the gaseous state. The decreases in pH decrease its toxicity due to conversion of ammonia into ammonium ion, which is much less toxic than the gaseous form.

Principal:
When nesselers reagent is added to a diluted ammonia solution, the liberated ammonia reacts with the reagent rapidly to form orange-brown product, which remains in colloidal

16

solution but flocculates on long standing. Visual comparison must be made before flocculation occurs.

Requirements:
Sample collection bottle Test tube Nesselers Reagent

Procedure:
Transfer 5ml of water sample in a test tube. Add 2-3 drops off Nesselers Reagent. Compare the colour with colour chart.

Observation:
AREA Ramnagar Suresh colony Cemetery road Haranganj Babugaon Pugmil Matwari Barabazar Aryanagar Anandpuri Distilled water WELL -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve SOURCE SUPPLY -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve BORING -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Result:
Ammonia was found to absent in sample.

Permissible limits Drinking water: should be absent Fresh water aquatic life 0.02mg/l
17

Ammonia is absent in water sample.

Precautions:
1. Nesselers Reagent is harmful; avoid contact with eyes& skin. 2. For protection, gloves could be used. 3. Do not delay more than2 mins to compare colour.

EXPERIMENT NO. -5
Aim: To test the hardness of water. Significance:
Hardness is the property of water, which prevents lather formation when using soap, and increases the boiling point of water. Hardness is principally due to the presence of calcium magnesium cations. However other cations such as strontium, iron and manganese also contribute to hardness.The anions responsible for hardness are mainly bicarbonate, carbonate, sulphate, chloride, nitrate and silicates etc.Hardness is called temporary if it is caused by carbonates and bicarbonates salts of the cations and since it can be removed simply by boiling the water. Permanent hardness is caused mainly by sulphates and chlorides of the metals. Hardness indicates water quality mainly as the sum of calcium and magnesium concentration both expressed as calcium carbonate in mg/l as follows. Soft Medium Hard Very hard 0- 75 mg/l 75- 150 mg/l 150- 300 mg/l > 300 mg/l

SOURCES:

18

Natural sources principally limestone, which is dissolved by percolating rainwater. Industrial and industrially related sources include the inorganic chemical industry and discharge form operating and abandoned mines.

Effects:
Hardness has no known adverse effects on health. However, some evidence has been given to indicate its role in heart disease. It is undesirable due to the formation of scales in boilers and other heat exchange equipments. Hard water is also unsuitable for domestic use in washing, cleaning and laundering

Principal:
This method depends on the ability of EDTA or its disodium salt to form stable complexes with calcium and magnesium ions. When the dye eriochrome black-T is added to a solution containing calcium, magnesium ions form a red- wine complex. This solution is titrated with a standard solution pf EDTA, which extracts calcium and magnesium from dye complex and the dye is changed back to its original blue colour. Eriochrome black-T is used to indicate the end point for the titration of calcium and magnesium together.

Requirements:
Apparatus required Test tube Sample collection bottle Micropipette

Chemical required Eriochrome black-T EDTA Ammonia buffer

Procedure:
Transfer 5ml of water sample in the test tube.

19

 Add 1-2 drops of ammonium buffer to raise the pH to 10. Add a pinch of eriochrome black-T powder. Take 1ml of EDTA solution in 1ml micropipette without any air bubbles. Titrate till colour changes from wine red to blue. Amount of EDTA consumed should be noted to calculate the value of hardness in water.

Observation:
PLACE Anandpuri Aryanagar Babugaun Bada Bazar Cemetery Road Haranganj Matwari Pugmil Ramnagar Suresh Colony SOURCE Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well EDTA (l) 500 500 250 650 560 550 500 500 350 500 300 400 700 600 250 650 650 250 600 600 600 550 350 450 550 300 400 600

20

Lab

Boring Supply Distilled Water

1300 250 50

Calculation:
Hardness as CaCO3 = (ml of EDTA consumed * 400) mg/l EDTA EDTA PLACE SOURCE consumed consumed(ml) (l) 0.5 Anandpuri Well 500 Boring 500 0.25 Supply 250 0.5 Aryanagar Well Boring Supply 650 560 550 0.65 0.55 0.56 Calculation 0.5* 400 = 200mg/l 0.25* 400 = 100mg/l 0.5* 400 = 200mg/l 0.65 * 400 = 260mg/l 0.55 * 400 = 220mg/l 0.56 * 400 = 224mg/l 0.5* 400 = 200mg/l .35* 400 = 140mg/l 0 0.5* 400 = 200mg/l 0.5 * 400 = 200mg/l 0.4 * 400 = 160mg/l 0.3 * 400 = 120mg/l 0.7 * 400 = 280mg/l 0.25 * 400 = 100mg/l 0.6 * 400 = 240mg/l 0.65 * 400 = 260mg/l 0.25 * 400 =

Babugaun

Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply

500 500 350 500 300 400 700 600 250 650 650 250

0.5 0.5 0.35 0.5 0.4 0.3 0.7 0.25 0.6 0.65 0.25 0.65

Bada Bazar

Cemetery Road

Haranganj

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100mg/l 0.65 * 400 = 260mg/l Matwari Well Boring Supply Well Boring Supply 600 600 600 550 350 450 0.6 0.6 0.6 0.55 0.45 0.35 0.6* 400 =240mg/l 0.6* 400 =240mg/l 0.6* 400 = 240mg/l 0.55 * 400 = 220mg/l 0.45 * 400 = 180mg/l 0.35* 400 = 140mg/l 0.55* 400 = 220mg/l 0.3 * 400 = 120mg/l 0.4* 400 = 160mg/l 0.6 * 400 = 240mg/l 0.25 * 400 = 100mg/l 1.300 * 400 = 520mg/l

Pugmil

Ramnagar Suresh Colony

Well Boring Supply Well Boring Supply

550 300 400 600 1300 250

0.55 0.3 0.4 0.6 0.25 1.300

Result:
Drinking water = 300 600 mg/l Maximum hardness found in boring water of Suresh colony i.e 520mg/l while minimum in distilled water i.e 20 mg/l. Water is potable for drinking.

Precautions:
Ammonia must be kept with tight lid otherwise, it may loose its capacity to raise the pH of the sample to 10. This whole test should be completed in 5 minutes, as there is high chance of precipitation of calcium as CaCO3 with lapse of time.

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PHYSICAL TESTS
23

EXPERIMENT NO. -6
Aim: To determine the pH of water.

Significance:
pH value gives an idea of the intensity of the acidic or basic nature of a water sample. pH scale for aqueous solutions lies between 0 14. The abundance of Hydrogen ion is what determines the acidity of a solution. More the hydrogen ions, the more acidic the sample is. Higher concentration of hydrogen ions gives higher score on the pH scale. Water acquires acidic or to alkaline property depending on the type of substances that is comes in contact with. This happens naturally or is sometimes man-induced. Rain water absorbs atmospheric carbon dioxide and becomes slightly acidic due to formation of carbonic acid. Therefore pH of natural rain water shows a range of 4.5 to 5.6.

Effects:
A pH below 4.0 gives a sour taste above 8.0 an alkaline taste. Water having higher values of pH increases scale formation in water heating apparatus. Disinfectant capacity of chlorine reduces if water is alkaline. pH below 6.5 cause corrosion in the pipes thereby releasing toxic metals such as Zinc, Lead, Cadmium, Copper, etc.In water treatment

24

systems, the dosage of alum is fixed according to the pH of the water. A pH raise in large lakes and reservoirs with excessive algae causes fish kills.

Principal:
pH paper turns red with acidic samples.

Requirements:
pH meter or ph paper. Sample collection bottle.

Procedure:
Take sample collection bottle and dip pH meter into it. Record corresponding pH value.

Observations:
PLACE Anandpuri Aryanagar Babugaun Bada Bazar Cemetery Road Haranganj Matwari SOURCE Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring Supply Well Boring 25 pH VALUE 7.62 7.82 8.47 7.59 8.09 7.78 7.88 7.12 7.82 7.42 7.68 7.88 7.80 7.41 8.47 7.42 7.39 8.47 7.14 7.34

Pugmil Ramnagar Suresh Colony Lab

Supply Well Boring Supply Well Boring Supply Well Boring Supply Distilled Water

7.33 7.49 7.26 7.14 7.85 7.31 7.20 7.67 7.40 7.40 6.5

Results:
The pH of water was found to be slightly basic.

Permissible limits
Potable water = 6.5 8.5 Irrigation water = 4.5 9.0 Fresh water (aquatic life) = 6.5 9 Marine water (aquatic life) = 6.5 8.5

Maximum pH in supply water of Anandpuri, Haranganj and cemetery road i.e. 8.47 while minimum in distilled water i.e. 7. Water is potable for drinking.

26

BIOLOGICA L TESTS

27

EXPERIMENT NO. 7
Aim: To determine dissolved oxygen (DO) of water. Theory:
Dissolved oxygen of water is readily available for aquatic live. The presence of DO water may be mainly attributed to two distinct phenomena: (i) direct diffusion from air; (ii) photosynthetic evolution by aquatic autotrophs. The estimation of DO is done by the titrimetric method. The oxygen of the water combines with manganous hydroxide, which on acidification liberates iodine equivalent to that of oxygen fixed. The iodine is titrated by standard sodium thiosulfate solution using starch as indicator, MnSO4 + 2KOH Mn (OH) 2+ O MnO (OH) 2+2H2SO4 Mn (OH) 2+K2SO4 MnO (OH) 2 MnSO4+K2SO4+3H2O+I2

The biochemical oxygen demand (BOD) is a way of expressing the amount of organic compounds in water as measured by the volume of oxygen required by bacteria to metabolize it under aerobic conditions. If the amount of organic matter in water is more, the more oxygen will be utilized by bacteria to degrade it.

Requirements:
Water sample Allylthiourea solution (0.5%) Sulfuric acid (1N). Sodium hydroxide (1N) Sodium thiosulphate (0.025) Manganous sulphate sol Alkaline iodine-azide sol Starch indicator BOD bottles Flask Pippette BOD incubater

28

pH meter.

Procedure:
A)

Preparation of chemicals:-

Alkaline iodine-azide solution : Add 100 gm of KOH and 50 gm of KI in 200 ml of distilled water. Manganese sulphate solution ( MnSO4. 4H2O) solution: Dissolve 100 gm of MnSO4.4H2O in 200 ml of distilled water, filter and keep. Starch indicator: Dissolve 5 gm of starch in 100 ml hot distilled water and a few drops of formaldehyde. Na- thiosulphate 0.025N

B) Titration procedure

Collect water sample and adjust the pH to neutrality. Fill the water sample in 2 BOD bottles of 300ml without bubbling. Add 2ml each of MnSO4 and alkaline iodide azide sol. In succession in 1 BOD bottle. Shake the bottle in the upside down direction. Add 2ml of H2SO4 acid and shake to dissolve the brown ppt. Take 50ml sample in flask and titrate with thiosulphate sol till the colour change to pale yellow Add 2 drops of starch sol to the above sol to change the colour to blue Titrate again with thiosulphate sol and v1 is noted Another bottle of same sample is incubated after adding 1 ml of allylthiourea for 3 days at 270C Then again titrate the sample as mentioned above and v2 is noted

29

Calculation:
From the noted v1 and v2 for each sample we calculate the DO for each value

DO (in mg\l) = [ (8*1000N) v] V

Where, V = vol of sample taken (ml) v = vol of titrant used N = normality of titrant * = 8 is constant since 1 ml of 0.025 sodium thiosulphate sol is equivalent to 0.2 mg of oxygen

Result:
The BOD of water sample in mg\l is calculated by the formula

BOD in mg\l = D1 D2

Precautions:
Bubbling in BOD bottle should be avoided . Separate pippetes foe different chemical should be used. Concentrated acids should be handled carefully

30

PLACE 1. Ramnagar 2. Babugaun 3. Matwari 4. Anandpuri 5. Cemetery road 6. Haranganj 7. Aryanagar 8. Suresh colony 9. Pugmil 10. Barabazar

SOURCE Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply Well Boring supply

D1-D2 (mg\l) 0.4 0.32 1.04 0.16 0.08 0.48 0.32 0.96 0.32 0.24 0.56 0.16 0.16 0.16 0.16 0.32 0.16 0.16 0.40 0.24 0.08 0.96 0.24 0.16 0.16 0.40 0.08 1.2 0.40 0.24

31

EXPERIMENT NO. 8
Aim: Bacteriological examination of water by multipletube fermentation test or multiple-test tube test.
Theory:
Multiple-Tube Fermentation Test or Most Probable Number (MPN) Test is the most often used technique for the sanitary analysis of water. The test is used to detect coliforms that make up approximately 10% of the intestinal microorganisms of humans and other animals and have found widespread use as indicator organisms of fecal contamination. The test is performed sequentially in three stages: presumptive, confirmed and completed test. Lactose broth tubes are inoculated with different water volumes in the presumptive test. Tubes that are positive for gas production are streaked on Endo agar plate; presence of green sheen completes the second round of the test. Finally, the presence of E. coli is established by Grams staining.

MEDIA USED:
Endo agar

Peptone 10g Lactose 10 g Dipotassium hydrogen phosphate 3.5 g Sodium sulphite 2.5 g Basic fuchsin 0.4 g Agar 15 g Distilled water 1000 ml PH 7.5

Lactose fermentation broth 1X & 2X

Lactose 5 g Peptone 5 g

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Beef extract 3 g Distilled water 1000 ml PH 6.9

NOTE: For 2X broth, double the quantity of all the ingredients, except the distilled water.

Mac Conkeys agar

Peptone 20 g Sodium chloride 5g Bile salt 1.5 g Lactose 10 g Neutral red solution (1% aqueous) 10 ml Crystal violet 0.001 g Agar 13.5 g Distilled water 1000 ml pH 7.1

DCA

Agar 15 gm Lactose- 10 gm Sodium citrate 8.5 gm

33

Sodium thiosulphate 5.4 gm Ferric citrate 1 gm Sodium deoxychlorate 5 gm Beef extract 5 gm Peptone 5 gm Neutral red -0.02 gm pH- 7.3 at 25 c Distilled water -1000ml

34

1. PRESUMPTIVE COLIFORMS TEST

Theory:
The presumptive coliforms test is used to detect coliforms in water sample. In this test lactose, fermentation tubes are inoculated with different water volumes and production of acid and gas from the fermentation of lactose in any of the tubes is a presumptive evidence of coliforms in the water sample.

Requirements:

Water sample Lactose broth medium Durham tubes 10 ml double-strength lactose broth tubes (LB 2X) 5 ml single-strength lactose broth tubes (LB 1X) Sterile pipettes, one each of 10 ml, 1 ml and 0.1 ml capacity Bunsen burner/Spirit lamp Mechanical pipetting device. Glass marker.

Procedure:

Collect water sample.

Label 6 double-strength lactose broth tubes 10 and 5 single-strength broth tubes 1 another five tubes 0.1.

Mix the water sample by shaking.

Aseptically inoculate each 10 tubes (LB2X) with 10 ml of water sample using 10 ml sterile pipette.

35

Using a 1 ml pipette, aseptically inoculate the five tubes (LB1X) with 1 ml of water sample.

Using a 0.1 ml pipette, aseptically inoculate the five tubes (LB1X) with 0.1 ml of water sample.

Incubate all the tubes aerobically at 350C for 48 hours.

PLACE

SOURCE BORING WELL SUPPLY Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present Bubbles present

INCUBATION PERIOD

OBSERVATION

Anandpuri Aryanagar Babugaun Bada bazaar Cemetery road Haranganj Matwari Pugmil Ramnagar Suresh colony Lab (Distilled water)

Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles Bubbles present present Bubbles absent

May be coliforms May be coliforms May be coliforms 24 hrs May be coliforms May be coliforms May be coliforms May be coliforms May be coliforms May be coliforms May be coliforms 48 hrs Coliforms absent

PRECAUTIONS:

36

 After sterilization, the Durham tubes in lactose fermentation tubes should be free from air bubbles.

37

2. CONFIRMED COLIFORMS TEST:

THEORY:
The test is used to confirm the presence of coliforms in water samples showing positive or doubtful presumptive test. In the confirmed test, water sample from all the positive presumptive lactose broth tubes were streaked on Endo agar plates.

REQUIREMENTS:
Endo agar plated Petri plates Positive lactose broth tubes LAF Inoculation loop Bunsen burner.

PROCEDURE:
Inoculate Endo agar plated Petri plates with the inoculums from all lactose broth positive presumptive tubes.

Incubate all the inoculated tubes at 350C for 48 hours.

Observation:
PLACE
Anandpuri Aryanagar Babugaun Bara bazaar Cemetery Haranganj Matwari Pugmil Ramnagar
Suresh colony

SOURCE BORING Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive WELL Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive SUPLLY Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

INCUBATION PERIOD

OBSERVATION
Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed Coliforms confirmed

24 hrs

38

D.W

Absent

Coliforms absent

39

I.
THEORY:

COMPLETED COLIFORMS TEST:

Completed test is used to establish the presence of coliforms bacteria and as a confirmatory test for the presence of E coli in a water sample. In the completed test, the samples from the positive Endo agar plates from the confirmed test are streaked onto a selective differential medium for coliforms and incubated into lactose broth tube as well as streaked on a nutrient agar plate to perform Gram staining. The medium commonly used is Endo agar i.e. selective in nature because of the dye basic fuchsin, which inhibits the growth of Gram-positive bacteria, allowing the growth of Gram-negative bacteria. Endo agar is differential in nature in that lactose-fermenting bacteria gives colored colonies (a positive confirmed test) due to the formation of a complex in Endo agar that precipitates out onto the coliforms colonies. Non-lactose fermenters produce colorless colonies on Endo agar agar. If there is production of acid and gas in the incubated lactose broth and there are rod shaped bacteria showing Gram-negative reaction, these confirm the presence of E coli in the water sample and is considered a positive completed test.

REQUIREMENTS:
Endo agar plates 24 hours coliform positive lactose broth Nutrient agar slant Inoculating loop Bunsen burner Grams Iodine Crystal violet Safranin Alcohol (50%) Distilled water Slide Microscope

PROCEDURE:

40

Streak the Mac Conkey agar plates from positive tubes with a sterile inoculating needle.

Incubate the inoculated plates for 24 hours at 350C in an inverted position.

Observe the plates after 24 hrs incubation for coliform colonies.

Prepare a thin smear of coliform colonies from positive Mac Conkey agar plates. Stain the slides with Gram-stain. Observe the slides under microscope for Gram-negative coliforms.

Gram staining Procedure:

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Make a slide of tissue or body fluid that is to be stained. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. Add the primary stain crystal violet and wait 1 minute. Rinse gently with water. This step colors all cells violet. Add Gram's iodine, for 30 seconds. It is not a stain; it is a mordant. It doesn't give color directly to the bacteria but it fixes the crystal violet to the bacterial cell wall. All cells remain violet. Wash with ethanol and acetone, the decolorizer. If the bacteria is Grampositive it will retain the primary stain. If it is Gram-negative it will lose the primary stain and appear colorless. Add the secondary stain, safranin, and wait 1 min, then wash with water for a maximum of 5 seconds. If the bacteria is Gram-positive then the cell will retain the primary stain, will not take the secondary stain, and will appear black-violet. If the bacteria is Gram-negative then the cell will lose the primary stain, take secondary stain, and will appear red-pink. If you have trouble remembering the order just remember the mnemonic:Come In And Stain. The C is for crystal violet, I is for iodine, A is for alcohol, and S is for safranin. Crystal violet is 2 g of 90% crystal violet dissolved in 20 ml of 95% ethanol. Gram's iodine is 1 g of iodine, 2 g of potassium iodide, dissolved in 300 ml of distilled water. Decolorizer is 50% ethyl alcohol, 50% acetone.

Observations: Test for presence of E.coli

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PLACE Anandpuri

SOURCE
Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply

OBSERVATION
E .coli absent E. coli present E .coli present E .coli present E .coli present

Aryanagar

E .coli present
E .coli absent E .coli present E. coli present E .coli absent, E. coli absent E. coli absent E .coli present E .coli present

Babugaun

Bada bazaar

Cemetery road

E. coli present
E .coli present E .coli present

Haranganj

E .coli present
E .coli present E. coli present

Matwari

E .coli present
E .coli present E .coli present

Pugmil

E. coli present
E coli present E coli present

Ramnagar

E coli present
E coli present E coli present

Suresh colony

E coli present

Test for presence of Salmonella typhi

Media utilized-------D.C.A
PLACE SOURCE OBSERVATION

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Anandpuri Aryanagar

Babugaun Bada bazaar Cemetery road Haranganj Matwari Pugmil Ramnagar Suresh colony Distilled water

Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply Boring Well supply

-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve +ve -ve +ve +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve +ve +ve -ve -ve -ve -ve -ve -ve -ve

RESULT:
Production of gas from lactose, presence of Gram-negative, non-spore forming rods result a positive completed test and confirmed the detection of E.coli bacteria in the water sample, indicating the fecal contamination of water, thus that water be considered as nonpotable.

CONCLUSION
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After performing different types of Physical, Chemical and Biological tests of the water sample from various sources of Hazaribag district, it was found that the physical and chemical properties of the water are within the prescribed limits, but when the biological tests were performed it was found that it has high level of fecal contamination , and some samples were also found to be contaminated with pathogenic microbes (Salmonella.typhi), so the water of Hazaribag is not potable and people of the town are at a high risk of waterborne diseases.

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