CHAPTER-III - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/12734/7/07... · 2015-12-04 · History: The city origins can ... and also for navigation. As a result, a number

CHAPTER-III

MATERIALS AND METHODS

STUDY AREA

History:

The city origins can be traced back to the rule of the king Raja Raja Narendra

who reigned around 1022 AD, after whom it was named Rajamahendri or

Rajamahendravaram. Remains of the 11th century fort walls & palaces still exist.

Rajamahendravaram was renamed as Rajahmundry during the British Rule.

Rajahmundry was claimed as the birth place of telugu language due to the city-born

poet, Nannayya who was also called Adi-Kavi (The first poet) of Telugu.

Rajahmundry was the hotbed of several movements during India’s freedom struggle

& acted as a base for many key leaders.

Demographics:

As of 2011 Indian Census, Rajahmundry had an urban population of

4,13,648 with the male : female ratio almost equal. The average literacy rate of

Rajahmundry is 72.50%, which is lower than the national average of 74.04%. Male

literacy is 19.44% & female literacy is 4.65%

Climate:

The Climate is hot & humid with mostly a tropical Climate. The mean

maximum temperature seems to be 360C. The hottest season is from April to June

with temperature ranging from 340C to 480C. The coolest months are December and

January with temperature ranging from 270C to 300C.There is a lot of rain due to the

monsoon & cyclonic storms in the Bay of Bengal.

Trade:

Rajahmundry is a Business Centre right since ages & acting as a Commercial

hub for both east & west Godavari Districts. It is a well Known Center for Cloth &

Bullion Business. Rajahmundry’s Cloth Market is one of the largest markets in India

with Many Co-operative handloom houses providing fine quality sarees as well as

men’s wear. After Visakhapatnam, the real estate has taken a leap in Rajahmundry.

Transportation:

Rajahmundry is well connected to all parts of the state. NH-5 passes through

Rajahmundry. With One National Highway & Two State Highways, it has excellent

Road Connectivity to all important places. It is the main transportation hub for both

the Godavari Districts. The City is also well connected by the railway network.

Rajahmundry Railway Station is one of the top revenue generators for South Central

Railways. Rajahmundry Airport, situated near Madhurapudi 18 Km away from the

heart of the city is accessible by air from many cities. The Recent Tourism

developments by government has made excellent platform for Rajahmundry to boast

itself as National Water-Way transport hub. The Waterway of Rajahmundry is being

developed by the Inland Waterways Authority of India (IWAI) & is scheduled to be

completed by 2013.

River Godavari:

The Godavari Takes rank amongst the great rivers of India after the

Ganges and the Indus. Rising some seventy miles north-east of Bombay & only fifty

miles from the Arabian Sea, it runs in a generally south-eastern direction across the

peninsula, till after a course of nearly ninety nine miles, it falls into the Bay of

Bengal. Its broad surface is plentifully strewn with floating debris of all kinds, giants

of the forest, bamboos interlaced in wild confusion, jungle grasses & river rushes

which the flood has deeply submerged or entirely washed away. Once every twelve

years a feast called Pushkaram is held on the banks of river Godavari. Crowds gather

on the morning, noon & night & convert the most favored resort in to huge standing

camps for the twelve days during which the festival lasts.

But while nature was thus too lavish of her beauties & the stream was so

scared, there was much suffering in the land due to pollution of the water. Being a

holy river, most of the religious activities are performed on the bank of river

Godavari. The great piece of work done by Sir Arthur Cotton by building Godavari

anicut is quiet amazing and appreciable.

Godavari Anicut (Barrage):

The construction of the anicut across the Godavari is a great boon to this part

of the country. Since its construction, the district is intersected with canals, useful not

only for purposes of agriculture, and also for navigation. As a result, a number of

irrigation channels and paddy transplantation has immensely increased. The Godavari

anicut is perhaps the noblest feat of engineering skill which has yet been

accomplished in British India.

Irrigation and Agriculture:

Godavari anicut is a gigantic barrier thrown across the river, in order to arrest

the unprofitable progress of its waters to the sea, and to spread them over the surface

of the country, thus irrigating copiously land which has hither to been dependent on

tanks or on the fitful supply of water from the river. The cultivators are highly

benefited because of the waters of Godavari. Large tracks of land, which had hitherto

been left arid and desolate, were thus reached and fertilized by innumerable streams

and channels. Irrigation pays everywhere, for without it millions could not live, and

millions more could be decimated by famine every few years. Reckoning its influence

upon railways, commerce and government of the country, its value is simply

inestimable.

Dependence of people on water:

In general, people of RJY are supplied with treated municipal water from river

Godavari. Many people even depend on underground water which is collected

through Bore wells. Usage of open wells is very rare now a days. Quiet a number of

people used packaged drinking water from home deliveries (Tins). Most of the houses

are even having household water filters to reduce water-borne infections.

MICROBIAL METHODOLOGY

Scrupulous care in the collection of samples for bacteriological examination

was taken to avoid accidental contamination of the sample during collection.

Sample containers: Sterilized glass bottles or screw capped tubes were taken

for sample collection. When samples of chlorinated water were taken, the content of

residual chlorine was determined at the sampling point itself.

Sampling procedures: The sampling bottle was kept unopened until the

moment at which it was required for filling. During sampling the stopper and neck of

the bottle were protected from contamination.

Preservation and storage of samples: The bacteriological examination of

water samples was initiated immediately after collection. The technical procedures for

bacteriological examination had started within one hour after collection.

For the isolation of fecal coliforms Membrane Filtration Technique was used.

Enteric bacteria isolated on respective selective or differential media were identified

on the basis of their colonial, morphological and biochemical characteristics

following Bergy’s Manual of Determinative Bacteriology, 1994.

Heterotrophic Bacteria:

Nutrient Agar Medium(5.0g Peptone 3.0g Beef extract, 5.0g Sodium Chloride,

15.0 Agar, Distilled water 1.0 liter, pH 7.0) was used for the isolation of various

Heterotrophic bacteria. The medium was prepared as per the composition, sterilized in

an autoclave at 15lbs pressure for 15 minutes and dispensed into sterile petri dishes.

Serial dilution of the test water samples was made with sterile distilled water.0.1ml of

the test samples from 103and 104 dilutions was dispensed into the petri dishes with

sterile nutrient agar medium. Plates were incubated at 37c for 36 hours. Analysis was

performed in triplicate. Enumeration of developed colonies was carried out on Quebec

colony counter.

Total Coliforms:

Endo Agar Medium [10.0g Peptone, 10.0g lactose, 3.0g sodium chloride,3.5g

dipotassium phosphate, 2.5g sodium sulphite, 0.4g Basic fuchsin (pararosaline), 15.0

Agar, distilled water 1.0liter, pH7.5] was used for the isolation of total coliforms.

Medium was prepared as per the composition, sterilized in an autoclave at 15lbs

pressure for 15minutes and dispersed into sterile petri dishes. Serial dilution of the test

water samples was made in sterile distilled water. 0.1ml of the test samples from

103and 104 dilutions was dispensed into sterile petri dishes with solidified medium.

The sample was spread on the medium by using sterile glass spreader to ensure

uniform distribution. Plates were incubated at 37oc for 36 hours. Analysis was

performed in triplicate. Enumeration of the colonies developed was carried out on

Quebec Colony Counter.

Faecal Streptococci:

Faecal streptococcal medium (6.0g beef extract, 10.0g peptone, 10g lactose,

0.4g sodium azide, 15.0g agar, distilled water 1.0 liter, pH 7.0) was used for the

isolation of faecal streptococci. Medium was prepared as per the composition,

sterilized in an autoclave at 15lbs pressure for 15minutes and dispersed into sterile

petri dishes. Serial dilution of the test water samples was made in sterile distilled

water. 0.1ml of the test samples from 103and 104 dilutions was dispensed into sterile

petri dishes with solidified medium. The sample was spread on the medium by using

sterile glass spreader to ensure uniform distribution. Plates were incubated at 37oC for

36 hours. Analysis was performed in triplicate. Enumeration of the colonies

developed was carried out on Quebec Colony Counter.

Faecal Coliforms:

Faecal coliform medium ( 5.0g proteose peptone, 3.0g yeast extract, 1.0g

sodium lauryl sulphate, 0.3g bromothymol blue, 15.0 g Agar, distilled water 11.0 liter,

pH7.3) was used for the isolation of faecal coliforms. Medium was prepared as per the

composition, sterilized in an autoclave at 15lbs pressure for 15minutes and dispersed

into sterile petri dishes. Serial dilution of the test water samples was made in sterile

distilled water. 0.1ml of the test samples from 103and 104 dilutions was dispensed into

sterile petri dishes with solidified medium. The sample was spread on the medium by

using sterile glass spreader to ensure uniform distribution. Plates were incubated at

37oC for 36 hours. Analysis was performed in triplicate. Enumeration of the colonies

developed was carried out on Quebec Colony Counter.

Staphylococci:

Mannitol salt agar (10.0g Mannitol, 10g peptone, 75.0g sodium chloride, 1.0g

beef extract, 0.025g phenol red, 15.0g agar, 1.0 liter distilled water, pH 7.4) was used

for the isolation of staphylococci. Medium was prepared as per the composition,

sterilized in an autoclave at 15lbs pressure for 15minutes and dispersed into sterile

petri dishes. Serial dilution of the test water samples was made in sterile distilled

water. 0.1ml of the test samples from 103 and 104 dilutions was dispensed into sterile

petri dishes with solidified medium. The sample was spread on the medium by using

sterile glass spreader to ensure uniform distribution. Plates were incubated at 37oC for

36 hours. Analysis was performed in triplicate. Enumeration of the colonies

developed was carried out on Quebec Colony Counter.

Useful for the selective isolation of pathogenic staphylococci, since most other

bacteria are inhibited by the high salt concentration colonies of pathogenic

staphylococci, are surrounded by a yellow halo, indicating mannitol fermentation. In

the above isolations, the number of bacteria were counted using the formula.

Total no. of bacteria present in 100 ml of sample = Number of colonies Amount plated X Dilution

Confirmation Tests For Different Bacterial Isolates:

Escherichia coli:

EMB Agar (10g peptone, 5g lactose, 2g dipottassium hydrogen phosphate,

0.4g eosin y, 0.065g methylene blue, 15g agar, 1.0L distilled water, pH 7.2) was used

for the confirmation of E.coli. The medium was prepared as per the composition and

sterilized at 15 lbs pressure for 15 minutes and dispensed into sterile petri dishes. The

inoculum was inoculated on the agar and plates were incubated at 370C for 36 hours.

Lactose and dyes eosin and methylene blue enable the identification of the colon

bacillus Escherichia coli. E.coli colonies are blue-black with a metallic green sheen

caused by the production of large quantity of acid.

Klebsiella pneumoniae:

Mac Conkey Agar (17g bacto peptone, 3g proteose peptone, 10g lactose, 15g

agar, 0.001g crystal violet, 0.03g neutral red, 1.0 L distilled water, pH 7.1) was used

for the confirmation of Klebsiella pneumonia. The medium was prepared as per the

composition and sterilized at 15 lbs pressure for 15 minutes and dispensed into sterile

petri dishes. The inoculum was inoculated on the agar and plates were incubated at

370C for 36 hours.

Salmonella typhi:

SS Agar (5g peptone, 5g beef extract, 10g lactose, 8.5g bile salts, 10g sodium citrate,

8.5g sodium thiosulfate, 1g ferric citrate, 0.0003g brilliant green, 0.025g neutral red,

15g agar, 1.0L distilled water, pH 7.0) was prepared as per the composition, sterilized

in an autoclave at 15lbs pressure for 15 minutes and dispensed into sterile petri dishes.

Inoculum was added to the medium and incubated at 370C for 24-48 hours.

Enterobacter species:

Violet Bile Glucose Agar (7.0g peptone, 3g yeast agar, 5g sodium chloride,

1.5g bile salts, 10g glucose, 0.03g neutral red, 0.002g crystal violet, 15g agar, 1.0 L

distilled water, pH 7.4) was used for the growth of Enterobacter species. The medium

was prepared as per the composition and sterilized at 15 lbs pressure for 15 minutes

and dispensed into sterile petri dishes. The inoculum was inoculated on the agar and

plates were incubated at 370C for 36 hours.

Vibrio species:

TCBS Agar (10g peptone, 10g sodium citrate, 3g sodium cholate, 10g sodium

chloride, 5g oxgall, 20g sucrose, 5g yeast extract, 10g sodium thiosulfate, 1g ferric

citrate, 0.04g bromothymol blue, 15g agar, 1.0L distilled water, pH 8.8) was prepared

as per the composition, sterilized in an autoclave at 15lbs pressure for 15 minutes and

dispensed into sterile petri dishes. Inoculum was added to the medium and incubated

at 370C for 24-48 hours.

Proteus species:

Hektoen enteric agar (12g proteose peptone, 12g lactose, 3g yeast extract, 2g

salicin, 9g bile salts, 5g sodium chloride, 5g sodium thiosulfate, 1.5g ferric

ammonium citrate, 0.1g acid fuchsin, 0.065g bromothymol blue, 15g agar, i.0L

distilled water, pH 7.5) was prepared as per the composition, sterilized in an autoclave

at 15lbs pressure for 15 minutes and dispensed into sterile petri dishes. Inoculum was

added to the medium and incubated at 370C for 24-48 hours.

Enterococcus species:

Mac Conkey Agar (17g bacto peptone, 3g proteose peptone, 10g lactose, 15g

agar, 0.001g crystal violet, 0.03g neutral red, 1.0 L distilled water, pH 7.1) was used

for the confirmation of Klebsiella pneumonia. The medium was prepared as per the

composition and sterilized at 15 lbs pressure for 15 minutes and dispensed into sterile

petri dishes. The inoculum was inoculated on the agar and plates were incubated at

370C for 36 hours.

Pseudomonas species:

Pseudomonas agar (10g peptone, 1.5g Magnesium sulphate, 1.5g dipotassium

hydrogen phosphate, 15g agar, 10ml glycerol, 1.0L distilled water, pH 7.3) was

prepared as per the composition, sterilized in an autoclave at 15lbs pressure for 15

minutes and dispensed into sterile petri dishes. Inoculum was added to the medium

and incubated at 370C for 24-48 hours.

Aeromonas species:

Starch Ampicillin Agar (10g casein enzymic hydroltsate, 5g sodium chloride,

1g starch, 15g agar, 1.0L distilled water, pH 7.4) was prepared as per the composition,

sterilized in an autoclave at 15lbs pressure for 15 minutes and dispensed into sterile

petri dishes. Inoculum was added to the medium and incubated at 370C for 24-48

hours.

Yersinia species:

Yersinia Selective Agar (20g peptone, 2g yeast extract, 20g mannitol, 2g

pyruvate, 1g sodium chloride, 0.01g Magnesium sulphate, 0.05g sodium

deoxycholate, 0.03g neutral red, 0.001g crystal violet, 1.0L distilled water, pH 7.4)

was prepared as per the composition, sterilized in an autoclave at 15lbs pressure for

15 minutes and dispensed into sterile petri dishes. Inoculum was added to the medium

and incubated at 370C for 24-48 hours.

Shigella species:

SS Agar (5g peptone, 5g beef extract, 10g lactose, 8.5g bile salts, 10g sodium

citrate, 8.5g sodium thiosulfate, 1g ferric citrate, 0.0003g brilliant green, 0.025g

neutral red, 15g agar, 1.0L distilled water, pH 7.0) was prepared as per the

composition, sterilized in an autoclave at 15lbs pressure for 15 minutes and dispensed

into sterile petri dishes. Inoculum was added to the medium and incubated at 370C for

24-48 hours.

Table: 1 Microbes and Diseases Caused by them

S.No. Microbe Infection caused

1. Escherichia coli Diarrhea, Gastroenteritis

2. Enterobacter aerogenes Gastro-intestinal disorders

3. Klebsiella pneumoniae Pneumonia

4. Salmonella typhi Typhoid fever

5. Shigella dysenteriae Bacillary dysentery

6. Vibrio cholerae Cholera

7. Pseudomonas aeruginosa Pyogenic infections

8. Yersinia enterocolitica Gastro-intestinal illness

9. Proteus vulgaris Nosocomial infections

10. Aeromonas hydrophila Intestinal & Respiratory illness,septicemia

11. Enterococcus fecalis Gastro-intestinal disorders

BIOCHEMICAL TESTS

Biochemical tests were performed in the present study as per the

protocols detailed in the manual “Microbiology and Laboratory Manual “by

Cappuccino and Sherman (2004) sixth edition and “Experiments in Microbiology,

Plant pathology and Biotechnology “ by K. R. Aneja (2003) fourth edition.

Indole Test: Tryptophan broth (10.g tryptophan, 5.0g sodium chloride, 1.0 liter distilled

water) was prepared as per the composition and sterilized in an autoclave at 15lbs

pressure for 15minutes. Broth was dispensed approximately 4.0ml per tube. Tubes

were inoculated with inoculum and incubated at 37oC for 24 to 48 hours. After

incubation, 1ml Kovac’s reagent was added to each tube. Development of cherry red

colour on the top layer of the tube after addition of Kovac’s reagent indicates positive

test for indole. Absence of cherry red colour indicates negative test.

Methyl Red Test: MRVP Broth (7.0g peptone, 5.0g dextrose, 5.0g dipotassium phosphate, 1.0

liter distilled water, pH 7.2) was prepared as per the composition. 5.0ml of the broth

was dispensed into each tube and tubes were sterilized in an autoclave at 15 lbs

pressure for 15 minutes. The tubes were inoculated with the inoculum and incubated

at 37oC for 24-48 hours. After incubation, 5 drops of methyl red indicator was added

to each tube. If the medium remains red colour after addition of methyl red indicator it

indicates a positive test. If methyl red gets decolorized and turns yellow colour it

indicates a negative test.

Voges Proskauer Test: MRVP Broth (7.0g 0g peptone, 5.0g dextrose, 5.0g dipotassium phosphate,

1.0 liter distilled water, pH 7.2) was prepared as per the composition. 5.0ml of the

broth was dispensed into each tube and tubes were sterilized in an autoclave at 15 lbs

pressure for 15 minutes. The tubes were inoculated with the inoculum and incubated

at 37oC for 24-48 hours. After incubation, Baritts reagent was added to the tubes.

Development of pink colour in the medium indicates a positive test. No change in

colouration indicates a negative test.

Citrate Utilization Test: Simmon citrate medium ( 1.0g Ammonium dihydrogen phosphate, 1.0g

Dipotassium phosphate, 5.0g Sodium chloride, 2.0g sodium citrate, 0.2g magnesium

sulphate, 0.8g Bromothymol blue, 15.0g Agar, 1.0liter distilled water, pH 6.9) was

prepared as per the composition 4ml of. The medium was dispensed into each tube.

The tubes were sterilized in an autoclave at 15lbs pressure for 15minutes and prepare

the slants. The agar slants were inoculated with the inoculum and incubated at 37oC

for 24-48 hours. Change of the colour of the medium to blue colour indicates a

positive test and no colour change of the medium indicates a negative test.

Carbohydrate Fermentation Test: Phenol red broth (10.0g peptone, 1.0g beef extract, 5.0g sodium chloride,

0.018g phenol red, 1.0 liter distilled water) was prepared as per the composition. 5.0g

of carbohydrate ( glucose,lactose, maltose, galactose, mannitol, sucrose) was added to

the contents. Approximately 4ml of the medium was dispensed into each tube. The

tubes were sterilized in an autoclave at 15lbs pressure for 15minutes. The tubes were

inoculated with the inoculum and incubated at 37oC for 24-48 hours. After incubation,

change in colour of the medium to yellow colour indicates a positive test and no

change in the colour indicates a negative test.

Catalase Test:

Inoculum was picked with an inoculating needle and placed on a clean glass

side. A drop of hydrogen peroxide was placed on the organism with a dropper. The

appearance of bubbles of oxygen within one minute after the addition of H2O2

indicates a positive test. No bubbles indicates a negative test.

Gelatine Liquefaction Test: Gelatin medium (30.0g Gelatin,10.0g proteose peptone, 15.0g agar, 10.0 g

sodium chloride,1.0 litre distilled water, PH7.2 +/- 0.2) was used for the identification

of micro-organisms where Proteus vulgaris will show positive test & E.Coli will show

negative test. Medium was prepared as per the composition. 4ml of medium was

taken into screw cap tubes.The tubes were sterilized at 15 lbs pressure for 15 minutes

& medium was allowed to solidify. The inoculum was stabbed into the medium with

an inoculating loop. Tubes were incubated at 370C for 48 hours. After incubation,

tubes were placed in a refrigerator at 40C for 30 minutes. Inoculated tubes that remain

liquefied even after placed in a refrigerator shows positive test as it indicates

hydrolysis of gelatin. Tubes that remain solidified even after chilling shows a negative

test.

Urease Test: Urea agar (1.0g peptone, 5.0g sodium chloride, 2.0g Potassium dihydrogen

Phosphate, 15.0g agar, 1.0 litre distilled water, pH 6.8+/0.2) was prepared as per the

composition and sterilized in an autoclave at 15 lbs pressure for 15 minutes. Medium

was cooled at 500C. 1.0g glucose & 6.0 ml phenol red were added to the medium &

steamed for 1 hour & then 100ml urea was added. Mix well & dispense the medium

into sterile test tubes. Inoculum was added & tubes were incubated at 370C for

24hours. After incubation, positive tubes show deep pink colour. Negative tubes do

not show any color change.

Oxidase Test: Trypticase Soy agar (15.0g trypticase,5.0g soy peptone,5.0g sodium

chloride,15.0 g Agar,1.0 liter distilled water) was prepared as per the composition,

sterilized in an autoclave at 15 lbs pressure for 15mins & poured into sterile petri

dishes. Using sterile technique, make a single-line streak inoculation of the inoculums

on the agar surface. Plates were incubated at 370C for 24 to 48 hours. After

incubation, oxidase reagent was added. Change of colour to purple indicates positive

test and no colour change indicates negative test.

Phenylalanine deaminase test: Phenylalanine agar (3.0g yeast extract, 2.0g phenylalanine, 1.0g disodium

phosphate, 5.0g sodium chloride, 15.0g agar, 1.0 liter distilled water,pH 7.3 +/0.2)

was prepared as per the composition to differentiate proteus species from other

members of enterobacteriaceae as phenyl pyruvic acid was formed from

phenylalanine. Medium was dispensed into tubes & sterilized in an autoclave at 15 lbs

pressure for 15 minutes. Tubes were inoculated and incubated at 370C for 24 to 48

hours. After incubation, ferric chloride was added to each tube. Production of green

colour indicates a positive test.

Hydrogen Sulfide Test:

SIM (Sulfide Indole Motility) agar (30.0g peptone, 3.0g Beef extract, 0.2g

Ferrous ammonium sulfate, 0.025g sodium thiosulfate,3.0 agar,1.0 liter distilled

water) was prepared as per the composition, dispensed into test tubes & sterilized in

an autoclave at 15 lbs pressure for 15 minutes. The Inoculum was inoculated by

stabbing. Plates were incubated at 370C for 24-48 hours. Presence of black

colouration along the line of stabbing indicates a positive test and no colour change

indicates a negative test.

Starch Hydrolysis: Starch agar (20.0g starch, 5.0g peptone, 3.0g Beef extract, 15.0g agar, 1.0 liter

distilled water) was prepared as per the composition, sterilized in an autoclave at 15

lbs pressure for 15 minutes & poured into sterile petri dishes. After solidification, a

single streak of inoculum was made in the centre of the plate. Plates were incubated at

370C for 48 hours. After incubation, flood the surface of the plates with the iodine

solution. A Clear zone surrounding the colonies shows a positive starch hydrolysis &

dark blue colouration of the medium indicates a negative result.

Triple Sugar Iron (TSI) Agar:

(1) carbohydrate fermentation (2) gas production.

Carbohydrate fermentation: TSI slants contain three carbohydrates: glucose

(0.1%), sucrose (1%), and lactose (1.0%), as well as phenol red (pH indicator) and

peptones. Fermentation of the carbohydrates into acid end-products will result in a

yellow color on the slant and/or butt as the acids react with the phenol red. All

organisms will generally ferment glucose before fermenting lactose and/or sucrose.

Results in the slant and butt are reported as "A" for acid, (indicated by a yellow color)

and "Alk" for alkaline (indicated by a red or orange color).

Alk slant / Acid butt: Only glucose fermentation has occurred. Since glucose

is present in a 0.1% concentration, the small amount of acid produced by glucose

fermentation is rapidly oxidized on the slant, resulting in an alkaline reaction. In the

butt, the acid reaction is maintained because of reduced oxygen tension and slower

growth of the organisms.

Acid slant / Acid butt: Glucose fermentation plus lactose and/or sucrose

fermentation has occurred. The acid end-products have reacted with the phenol red in

both the slant and the butt.

Alk slant / Alk butt: No carbohydrate fermentation has occurred. Instead,

peptones are catabolized resulting in an alkaline pH due to the production of

ammonia.

Most probable number Methoed Presumptive test is specific for detection of coliform bacteria. In the lactose

medium tubes are inoculated 10ml, 1ml, 0.1ml aliquots of the water samples. The

series consists of three groups, each composed of three tubes of the specified medium.

Development of gas in any of the tubes is presumptive evidence of the presence of

coliform bacteria in the water samples.

Confirmed test requires that selective and differential media such as eosin-

methylene blue (EMB) or endo agar be streaked from a positive lactose broth or

MacConkey broth tube obtained from the presumptive test. In the presence of acid

environment, EMB forms a complex that precipitates out onto the coliform colonies,

producing dark centers and green metallic sheen. This reaction is characteristic for

Escherichia coli, the major indicator of fecal pollution.

Completed test is the final analysis of the water sample. It is used to examine

the coliform colonies that appeared on the EMB or endo agar plates used in the

confirmatory test plate and inoculated into a tube of lactose broth and inoculated on a

nutrient agar slant to perform a Gram stain.

Table:2 The most probable number (MPN) of coliforms Standard methods for the Examination of water and waste water, APHA, New York, 1998.

3 of 10ml each 3 of 1ml each 3 of 0.1ml each MPN Index per

100ml 0 0 1 3 0 1 0 3 1 0 0 4 1 0 1 7 1 1 0 7 1 1 1 1 1 2 0 1 2 0 0 9 2 0 1 14 2 1 0 15 2 1 1 20 2 2 0 21 3 0 0 23 3 0 1 39 3 0 2 64 3 1 0 43 3 1 1 75 3 1 2 120 3 2 0 93 3 2 1 150 3 2 2 210 3 3 0 240 3 3 1 460 3 3 2 1100 3 3 3 2400

PHYSICO-CHEMICAL METHODS

Water Sampling:

Water samples were collected during the three seasons from March 2009 to

February 2011 in the morning hours. Samples were collected into sterile tightly

capped glass bottles labeled with sample code and transported to the laboratory within

two hours in an ice box and stored at 40C. The samples were processed and analyzed.

Field Investigation:

The actual sampling locations in the water body were selected depending upon

the area, seasons, population, environment, etc. Accordingly, I have collected water

samples from different places of Rajahmundry Town which is located on the banks of

river Godavari. About 70 drinking water samples have been collected from various

sources such as domestic taps, public places, bore wells, open wells, minerals waters

and filter waters.

Experimental Details:

The present investigation was carried out by collection of drinking water

samples (bore water, well water, tap water and mineral water) from different areas.

The samples thus collected were analyzed for different parameters.

Collection of Water Samples:

Water samples were collected in sterile glass bottles (APHA, 1985). For

bacteriological analysis, samples of water were collected in pre- sterilized bottles and

preserved at 40C. The water temperature was recorded at the site using a sensitive

mercury thermometer and pH was measured in-situ using pH meter. For DO and BOD,

the samples were collected in glass stoppered bottle very carefully in order to avoid

contact of the sample with air. The bottles were completely filled with water. The

conductivity, TDS, turbidity, chlorides, total hardness, DO, BOD, COD, sulphate,

phosphate and nitrate were analyzed in the laboratory using standard methods.

Methods of analysis of water samples:

The methods suggested for drinking water analysis as per APHA (1985) have

been used. The standard methods of analysis for physical and chemical properties of

drinking water were used.

1. PH:

The PH of drinking water samples was determined by potentiometric method

as described by APHA (1985). pH is a term used to express the intensity of the acid or

alkaline condition of a solution. It is the expression of hydrogen ion concentration or

more precisely the hydrogen activity. pH is generally expressed in terms of negative

logarithm of hydrogen ion concentration i.e. by equation.

pH= - log [H+]

The pH determination is usually done by pH meter method that is the most

accurate method and free of interference. pH meters are usually standardized with

standard buffer solutions. PH 4 Buffer solution – Dissolve 1.012 gm anhydrous

potassium hydrogen phthalate KHC8H4O4 in D.W. and make up to 100ml in

volumetric flask.

PH 7 Buffer solution – Dissolve 1.361gm anhydrous potassium dihydrogen

phosphate KH2SO4 and 1.420 gm anhydrous disodium hydrogen phosphate

Na2HPO4 in D.W. and make upto 1000 ml in volumetric flask.

Measurement of PH

Follow the manufacturers instructions to operate the PH meter and determine

the PH of the sample of water. Before the measurement of PH of sample, Standardize

the PH meter using standard buffer solution near that of the sample to be tested. Check

the electrode response occasionally by measuring the PH of another standard buffer

solution with a differentPH.

2. Electrical conductivity:

Electrical conductivity is a measure of a waters capacity to convey electric

current. The ability of a solution to conduct an electric current is governed by the

migration of solutions and is dependent on the nature and number of the ionic species

in that solution. This property is called electrical conductivity. Electrical conductivity

of water is directly proportional to its dissolved mineral matter content. The unit of

conductivity is micro-siemens (µs/cm).

The conductivity of sample was determined with the help of digital

conductometer. The cell constant was determined by placing N/10 Kcal solution in

the given conductivity cell. Sample was taken in a clean beaker and conductivity cell

immersed in it and conductivity was measured.

3. Solids:

‘Solids’ is a term applied to all matter except the water contained in liquid

materials and thus definition of solids refers to the matters that remains as residue

upon evaporation and drying at a definite temperature. Total solids is the residue that

includes both dissolved solids and suspended solids. The amount and the nature of

dissolved and un-dissolved matter occurring in liquid material vary considerably.

Determination of dissolved and un-dissolved matter is made with filtered and

unfiltered portions of samples. The un-dissolved matter is usually referred to as

‘suspended solid’.

The common practice of determination of solids is to evaporate a suitable

volume of the sample in a dish and drying at 1050C followed by weighing. The

increase in weight represents the amount of solids. For evaporation, dishes of 50 to

250 ml capacity are employed. Dishes made up of various materials are available such

as platinum dish, porcelain dish, silica etc.

Total dissolved solids :

Centrifuge or filter a suitable volume of sample through whatman No. 30 or

equivalent filter paper. Evaporate the filtered sample in a dish. Dry the residue at 103-

1080C or 179- 1810C. The increase in weight of dish equals the total dissolved solids.

It may also be obtained by the difference between total solids and total suspended

solids. Express the result as total dissolved solids on drying at –0c in terms mg/L.

4. Total hardness:

Hardness is the capacity of water for reducing and destroying the lather soap.

Hardness in water is due to the natural accumulation of salts from contact with soil

and geological formations or it may enter from direct pollution by industrial effluents.

Calcium and magnesium are the principle cations causing hardness.

The term total hardness indicates the concentration of calcium and magnesium

ions only. The total hardness is expressed in terms of calcium carbonate. Among the

methods available for the determination of hardness, The EDTA titrimetric method is

precise one and can be performed rapidly.

Reagents :

1. Calcium standard solution- weigh accurately 1.0 gm of pure calcium carbonate and

place in a 250 ml conical flask using 50 ml d.w. Add 20 ml 1N Hcl. Warm until the

solution is complete. Cool and transfer to 1000 ml volumetric flask and make upto the

mark with D.W.

1 ml=1.0 mg CaCO3

2. Standard EDTA titrant 0.02N- weigh accurately 3.7239A.R. grade disodium ethylene

diamine tetra acetate dihydrate (ethylene diamine tetra acetic acid disodium salt

EDTA) and dissolve in D.W. and make upto 1000 ml.

1 ml of exactly 0.02 N EDTA = 1.0 mg CaCO3

3. Ammonia-Ammonium chloride buffer - Dissolve 16.9 gm ammonium chloride in 143

ml conc. ammonia solution. Add 1.25 gm magnesium salt of EDTA and dilute to 250

ml with D.W. Add this solution to 143 ml conc. ammonia solution in which 16.9 gm

NH4cl has already been dissolved. To attain highest accuracy, it has to be adjusted to

exact equivalence by appropriate addition of small amount of EDTA or MgSo4.

4. Sodium sulphide inhibitor- Dissolve 5 gm Na2S. 9H2O or 3.7 gm Na2S. 5H2O in 100

ml distilled water.

5. Eriochrome black T- indicator-

a) Mix 0.5 to 1 gm of the dye in 100 gm of triethanolamine.

b) Mix together 0.5 gm of the dye and 100 gm sodium chloride to prepare a dry powder

mix.

Procedure:

1. Place a suitable volume of sample in a conical flask and dilute to 50 ml.

For total hardness below 100 mg/L, use 100 ml sample.

For total hardness greater than 100 mg/L, use 50 ml sample.

For other, use 25 ml diluted to 50 ml.

Whatever be the volume of the sample taken, it should not consume more than 15 ml

EDTA titrant.

2. Add 1ml buffer solution per 50 ml volume. Add 1 ml sodium sulphide inhibitor if

necessary. Add 1 drop of indicator solution or an appropriate of dry powder.

3. Titrate with standard EDTA titrant solution slowly, until a reddish tinge appears and

add the last few drops within 3-5 seconds. At the end point, the solution will be blue.

Calculation :

Hardness as CaCO3 mg/L = ML EDTA titrant x E x 1000 ml sample taken for titration Where E is Mg CaCO3 equivalent to 1.0 ml EDTA.

Hardness as Ca mg/l = ml EDTA titrant x E x 1000x 0.4 ml sample for titration.

5. Calcium:

Calcium was determined by EDTA titrimetric method.

Reagents :

1. Sodium hydroxide 1 N – Dissolve 40 gmNaoH in about 100 ml d.w. and make

upto 100 ml.

2. Murexide indicator ( Ammonium purpurate indicator )- Prepared by dissolving 150

mg of the dye in 100 gm absolute ethylene glycol or by grinding 200 mg murexide

with 100 gm solid Nacl.

3. Standard EDTA titrant 0.02 N-(see in total hardness).

Procedure :

1. Place in a conical flask 50 ml of sample or a suitable aliquot that consumes not more

than 5 ml EDTA titrant. Dilute to 50 ml d.w.

2. Add 2 ml sodium hydroxide solution per 50 ml volume.

3. Add 1-2 gm of indicator if powder is used or 1-2 drops if a solution is used.

4. Titrate immediately with EDTA to proper end point.

Calculation :

If EDTA titrant is exactly 0.02 N

Mg/L calcium as CaCO3 = ml EDTA titrant x1x1000 ml sample taken for titration

Mg/L calcium as ca = ml EDTA titrant x 1x 0.4 ml sample taken for titration.

6. Chloride :

Chloride is the common anion found in water and sewage. The concentration

of chloride in natural water varies from few milligrams to several thousand milligrams

per liter. Higher concentration of chlorides may be due to contamination by sea water,

sewages or industrial effluents.

Chlorides were determined by silver nitrate method. Silver nitrate reacts with

chloride ions to form silver chloride. The completion of reaction is indicated by red

colour produced by the reaction of silver nitrate with potassium chromate which is

added as an indicator.

AgNo3 + Cl → AgCl + No3

2AgNo3 + K2Cro4 → Ag2Cro4 + 2KNo3

2AgNo3 + K2Cro4 → Ag2Cro4 + 2KNo3

Reagents :

1. Standard silver nitrate titrant 0.0282 N – Dissolve 4.791 gm silver nitrate in d.w. and

make upto 1000 ml in a volumetric flask. Standardise it against 0.0282 N sodium

chloride.

2. Standard sodium chloride titrant 0.0282 N – Dissolve 1.648 gmNacl and make upto

1000 ml in volumetric flask.

1.00 ml = 1.0 mg Cl

3. Potassium chromate indicator – Dissolve 25 gm potassium chromate K2CrO4 in 100

ml d.w.

Procedure :

Place 100 ml of sample or a suitable aliquot containing not more than 10mg.

chloride. Add 1 ml potassium chromate indicator solution. Titrate against standard

silver nitrate solution with constant stirring until a reddish coloration persist. Conduct

a blank by placing 100 ml chloride free distilled water instead of sample. Record the

reading.

Calculation :

Mg/L chloride as Cl- = (ml AgNo3 solution of sample – ml AgNo3 solution for blank) x Normality of AgNo3 x

35.45 x 1000

Ml sample taken

7. Dissolved oxygen :

Oxygen is dissolved in most waters in varying concentrations. Solubility of

oxygen depends on temperature, pressure and salinity of water. It is essential to the

life of fish and other aquatic organisms. Test for dissolved oxygen is generally not

carried out for unpolluted waters. This test is an indicator of the purity achieved

during treatments. Also it is the basis of BOD test.

Iodometric method is used for the determination of dissolved oxygen.

Probably winklers method is used to determine DO.

Apparatus :

Generally BOD bottles of 300 ml capacity are used.

Reagents:

1. Manganoussulphate solution – Dissolve 91.0 gmmanganoussulphate monohydrate

MnSO4. H2O in distilled water. Filter is necessary. Dilute to 250 ml. 1 ml of this

solution when treated with 50 ml of acidified potassium iodide solution should not

liberate iodine.

2. Alkali – iodine – azide reagent –

a. Dissolve 175 g potassium hydroxide and 37.5 gm potassium iodide in distilled water

and dilute to 250 ml.

b. Dissolve 2.5 gm sodium azide in 10 ml d.w. Pour the azide solution to alkali – iodide

solution and mix well.

3. Conc. sulphuric acid.

4. Phosphoric acid – 85 – 90 %

5. Sodium thiosulphate solution 0.1 N – Dissolve 24.82 gm sodium thiosulphate

Na2S2O3 .5H2O in boiled and cooled D.W. and make upto 1000 ml in a volumetric

flask.

6. Sodium thiosulphate solution 0.025 N – Dilute appropriate volume of 0.1 N sodium

thiosulphatesolution to 1000 ml D.W. in volumetric flask.

1.0 ml 0.025 N thiosulphate = 0.2 mg D.O.

7. Starch solution – Prepared by dissolving 1.5 gm starch soluble in 250 ml of D.W., boil

for few minutes and keep the solution overnight.

Procedure:

a. Fixation of dissolved oxygen –

Fixation of dissolved oxygen must be performed immediately after the samples has

been collected.

1. Take water samples in BOD bottles (of 300 ml capacity)

2. Remove the stopper from sample bottle. Add 1ml of mangnoussulphate solution by

inserting tip of pipette just below water surface.

3. Add 1 ml alkaline iodide solution in the same manner. Stopper the bottle without

entrainment of air and mix by inverting the bottle atleast about 10 times.

4. Allow the precipitate to settle completely leaving a clear supernatant liquid

5. Carefully remove the stopper and add 1 ml of conc. H2So4. Stopper the bottle and

mix thoroughly until dissolution is complete.

b. Titration –

Take 50 ml of sample in a flask and immediately titrate with 0.025 N sodium

thiosulphate to pale yellow colour. Now add 1 ml of starch indicator. Colour of

sample turns blue and titration continue till blue colour disappears. Record Reading.

Calculation:

Mg of O2 per liter = Used volume of titrant x 1000x 0.2. volume of sample

8. Biochemical oxygen demand :

Biochemical oxygen demand is a test of great value in the analysis of sewage

industrial effluents and grossly polluted drinking water. BOD refers to the quantity of

oxygen required by bacteria and other micro organisms in the biochemical

degradation and transformation of organic matter under aerobic conditions. The basic

principle underlying the BOD determination is the measurement of the dissolved

oxygen content of sample before and after 5 days incubation at 200C.

Reagents :

Same as like for D.O.

Procedure :

BOD of water sample collected was determined in the laboratory by following

procedure. Take water sample in BOD bottle. Remove stopper from sample bottle and

add 1 ml of MnSo4 soluion by inserting tip of pipette just below water surface. Add 1

ml of alkaline iodide solution as same manner. Mix the sample by inverting bottle

several times. Allow precipitate to settle down for few minutes. After setting of ppt.

clear fluid obtained in the upper portion of bottle. Take 50 ml of sample in which DO

is to be fixed in a flask and titrate with 0.025 N Na2S2O3 solution to form yellow

colour. Now add 1ml of starch solution as an indicator and titrate till blue colour

disappears. Record burette reading. Remaining water samples from BOD bottle were

incubated subsequently at 200c for 5 days. After 5 days, follow the same procedure

and titrate with 0.025 N Na2S2O3 solution and record burette reading and calculate

D.O.

Calculation :

BOD = D1- D2

Where D1 = Initial dissolve oxygen content.

D2 = Amount of oxygen after 5 days.

9. Sulphate :

Sulphate is one of the major anions occurring in natural waters. Sulphate

being a stable highly oxidized and soluble form of sulphur. It is generally presenting

natural surface and ground water. Sulphate may enter natural waters through

weathering of sulphide bearing rocks or by direct dissolution of evaporation deosits. It

may be leached from sedimentary rocks and particularly from sulphate deposits.

Effluent from certain industries may also be a major source of sulphate to the

receiving waters. Another significant source to water system is air borne industrial

pollutants containing oxides of sulphur which is converted to sulphuric acid in

precipitation (acid rain). ulphate can be determine by Turbidimetric method.

Reagents :

1. Conditioning reagent- Dissolve 75 gm sodium chloride in 300 ml distilled water. Add

30 ml conc. HCl and 100 ml 95% ethyl alcohol. Add 50 ml glycerol and mix well.

2. Barium chloride crystals.

3. Standard sulphate solution – Dissolve 147.9 mg anhydrous sodium sulphate in

distilled water and make up to 1000 ml.( 1.0 ml = 100 µg

So4 )

Procedure :

1. Measure 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 ml of standard sulphate solution

and dilute to 100 ml.

2. Add 5.0 ml conditioning reagent and mix well using magnetic stirrer.

3. While stirring, add 0.5 gm barium chloride crystals and continue to stir exactly one

minute.

4. Measure the optical density using a spectrophotometer at a wavelength of 420 nm.

5. Carry out a blank determination on the reagents used.

6. Measure a suitable quantity of sample and dilute to 100 ml, add 5.0 ml of

conditioning reagent, add 0.5 gm barium chloride crystals and precipitate is obtained.

7. From calibration graph, read the mg of sulphate equivalent to the optical density.

Mg sulphate as SO4-- = Mg SO4 x 1000

Ml sample taken for estimation .

10. Phosphate :

Phosphate may occur in surface or ground waters as a result of leaching from

minerals, from agricultural run off, as a result of industrial wastes and as a major

element of municipal sewage due to the utilization of synthetic detergents.

Phosphorous occurs in natural waters and waste waters in the form of phosphate. The

presence of phosphate in large quantities in fresh water indicates pollution through

sewage and industrial wastes. Though phosphate causes problems in surface waters,

its presence is necessary for biological degradation of wastewater. Phosphate is

determined by Stannous chloride method.

Reagents :

1. Phenolphthalein indicator

2. Sulphuric acid – nitric acid solution - Carefully add 75 ml conc. H2So4 to about 150

ml distilled water and cool. Add 1 ml conc. HNO3 and dilute to 250 ml with D.W.

3. Ammonium molybdate solution –

a) Dissolve 25 gm ammonium molybdate in about 200 ml D.W.

b) Add carefully 280 ml conc. H2So4 to 400 ml D.W. and cool.

Add the molybdate solution to diluted acid and dilute to 1000 ml.

4. Stannous chloride solution – Dissolve 2.5 gm stannous chloride in 100 ml glycerol

and heat in a water bath. Mix by stirring with glass rod.

5. Phosphate stock solution – Dissolve 439 mg anhydrous potassium dihydrogen

phosphate KH2Po4 in D.W. and make upto 1000 ml in volumetric flask.

1.0 ml = 100 µg

6. Phosphate standard solution – Pipette 10 ml phosphate stock solution into 1000 ml

volumetric flask and make upto the mark with distilled water.

1.0 ml = 1 µg

Procedure :

1. Place 100 ml or suitable aliquot of the sample containing not more than 20µg P in a

100 ml nessler tube. Add 1 drop of phenolphthalein indicator. If any pink colour

appears, destroy it by adding one or two drops of sulphuric – nitric acid solution.

2. Into a series of 100 ml nesslers tube , pipet appropriate volumes of phosphate working

solution covering the range upto 20g P. Dilute to 100 ml. Include a nessler tube

containing 100 ml distilled water as the blank.

3. To the blank, standard and sample, add 4.0 ml of ammonium molybdate solution and

0.5 ml stannous chloride solution. Mixing after each addition.

4. After 10 min. but before 12 min., measure the colour using a spectrophotometer at

690 nm.

5. Prepare a calibration curve and find out the number of microgram of P equivalent to

the observed optical density of the sample.

Express the result as Mg phosphate as P per liter of sample.

11. Carbonates and Bicarbonates:

Carbonates and Bicarbonates were determined titrimetrically as per Richards

(1954).

Reagents :

1. Phenolphthalein indicator: 0.25% solution in 60% ethyl alcohol

2. Methyl orange indicator: 0.5% solution in 95% alcohol

3. Standard sulphuric acid (0.02 N): Dilute 2.8 ml of this solution to again 1 litre for

getting 0.02 N H2SO4. Standardize the solution.

Method :

1. In a porcelain dish 5 ml of the water sample (containing not more than one

milliequivalent of carbonate plus bicarbonate) is diluted with distilled water to about

25 ml.

2. A pink colour produced with a few (2 to 3) drops of phenolphthalein indicates

presence of carbonate and it is titrated with 0.02 N sulphuric acid until the colour just

disappears (phenolphthalein end point ) because of alkali carbonate being converted

into bicarbonate. This burette reading is designated as b.

3. To the colourless solution from this titration, 1 to 2 drops of methyl orange or methyl

red indicator are added and the titration continued till the colour changes from yellow

to rose red.

4. Record the final reading which is marked as c.

Observations :

1. Volume of aliquot taken = ml

2. Burette reading (ml) = a b c

3. Volume of std. H2SO4 used for half neutralization of carbonate = (b- a) = x

4. Vol. of std. H2SO4 used for complete neutralization of carbonates = 2x

5. Vol. of std. H2SO4 used for complete neutralization of CO3 + HCO3 = c-a = y

6. Vol. of std. H2SO4 used for neutralization of HCO3 = y-2x

Calculations :

CO3-inmilliequivalent per litre = vol. of H2SO4 (2x) x N x 1000

ml of water sample

Where N = Normality of H2SO4.

HCO3- inmilliequivalent per litre = Normality of H2SO4 x Vol.of H2SO4 x 1000

ml of aliquot taken

HCO3- mg/liter = (y-2x) x 10.1 x 100

ml of aliquot taken

12. Nitrate – Nitrogen:

The amount of nitrate nitrogen in the sample is determined by phenol disulfonic acid

method.

Reagents :

1. Phenol disulfonic acid – Dissolve about 25 g of pure phenol in 150ml of conc.

H2SO4 and add 75 ml of fuming sulphuric acid. Heat for two hours over a boiling

water bath.

2. Sodium hydroxide (12 N) – Dissolve about 480 gm of pure sodium hydroxide in

distilled water and make upto about 1 liter.

3. Standard nitrate solution – Dissolve 0.7216 gm of pure potassium nitrate in 1 liter of

distilled water. Evaporate 50 ml of this solution to dryness on the water bath. Moisten

the residue with 2 ml of phenoldisulfonic acid rubbing it well into the residue to

insure intimate contact. Dilute to 500 ml with distilled water.

Procedure :

1. Filter 30 -35 ml of the sample through a filter paper.

2. Evaporate 25 ml. of the filtrate to dryness on water bath (use a smaller amount if

nitrate content is high.)

3. Moisten the residue with 1ml of phenoldisulfonic acid.

4. Dilute to about 20 ml with distilled water.

5. Add a 50 % solution of sodim hydroxide until the maximum yellow colour is

developed. (not more than 5-6 ml of NaoH will be required).

6. Filter into a 100 ml nessler tube, rinse the dish and paper with distilled water.

Add the filtered rinsing to the filtrate and make up to the mark with distilledwater.

7. If the permanent standards are available proceed to step no. 8 if not make up the

temporary standard by placing 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 ml. of

standard sodium nitrate solution in 100ml of nessler tube and adding 2 ml of 50 %

sodium hydroxide.

8. Dilute to the mark with distilled water.

9. Compare the colours and record the standards having a colour nearest to that of the

sample.

13. Sodium :

Sodium is present in most natural waters from negligible to appreciable

concentrations. It is of importance when salinity or total dissolved solids are a

consideration in the use of water.

It can be determined by flame photometer method. This method is by far the

simplest and most rapid method for determination of sodium. Even very small amount

of sodium in large proportion of water can be determined by this method.

Reagents:

1. Deionised distilled water – Prepared by passing D.W. through a mixed bed of ion-

exchange resins. Use only deionised distilled water for the preparation of solution and

dilutions.

2. Sodium stock solution – Dissolve 2.5422 g sodium chloride, NaCl( previously dried at

1400c for 1hr. ) in little deionised distilled water and make up to 1000 ml in a

volumetric flask.

1.0 ml = 1.0 mg Na.

Make dilutions as required for use and determine sodium content using flame photometer.

14. Potassium:

Potassium is determined by flame photometric method.

Prepare the potassium stock solution – Dissolve 1.907 gm potassium chloride, KCl

dried at 110oc in little de- ionized distilled water and make up to 1000 ml in a

volumetric flask.

1.0 ml = 1.0 mg K

Make dilutions as required for use with deionised water.

Determine the potassium content of the sample using flame photometer with usual

precautions. Express the result as Mg potassium as K per liter of the sample.

Table :3 Analytical methods and equipment used in the study

Sl.No. Parameter Method Instruments/Equipment A. Physico-chemical 1. pH Electrometric pH Meter 2. Conductivity Electrometric Conductivity Meter 3. TDS Electrometric Conductivity/TDS Meter 4. Nitrate Ultraviolet screening UV-VIS

Spectrophotometer 5. Chloride Titration by AgNO3 - 6. Sulphate Turbidimetric Turbidity Meter 7. Alkalinity Titration by H2SO4 - 8. Phosphate Molybdophosphoric acid UV-VIS

Spectrophotometer 9. Hardness Titration by EDTA - 10. Sodium Flame emission Flame Photometer 11. Potassium Flame emission Flame Photometer 12. Calcium Titration by EDTA - 13. Magnesium Titration by EDTA - 14. DO Titration by Sodium

thiosulphate solution -

15. BOD 5 days incubation at 20oC followed by titration

BOD Incubator

Table :4(a) Standards of bacteriological quality for drinking water

S.No Characteristics ISO WWHO

1 Total Coliform

MPN/ 100ml

<10 Nil

2 Fecal Colifrom

MPN/ 100ml

Absent Nil

Table:4(b)Standards of physical, chemical quality for drinking water

S.NO Parameter ISO(10500:2004)

(Desirable limit)

WHO (Maximum

allowable limit)

1 pH 6.5-8.5 6.5-8.5

2 Conductivity 300-400us/cm -

3 Total Alkalinity 200mg/L 120mg/L

4 Total dissolved solid 500mg/L -

5 Total Hardness 300mg/L -

6 Calcium 75mg/L 75mg/L

7 Magnesium 30mg/L 50mg/L

8 Nitrite - 3mg/L

9 Potassium - -

10 Sodium - 200mg/L

11 Fluoride 1mg/L 1mg/L

12 Chloride 250mg/L 250mg/L

13 Sulphate 200mg/L 500mg/L

14 Phosphate - -

15 DO - 5.0mg/L

16 BOD - -