Raw Water Testing (Method of Analysis) as per IP/BP/USP

1.0  Description

Clear, colorless, odorless and tasteless liquid.

2.0  pH

Apparatus: pH meter

Procedure:  Immerse the electrodes in the solution being examined and measure the pH.

Limit: Between 6.5 to 8.5

3.0  Heavy metals

Reagent: Acetate buffer pH 3.5, Thioacetamide

Test preparation: Heat 200 m in a glass-evaporating dish on a water-bath until the volume is reduced to 20 ml. To 12 ml of the prescribed aqueous solution add 2 ml of acetate buffer pH 3.5, mix, add 1.2 ml of thioacetamide reagent, mix immediately and allow to stand for 2 minutes.

Standard preparation: Take 10 ml of lead standard solution (1 ppm Pb), and 2 ml of the solution being examined. Add 2 ml of acetate buffer pH 3.5, mix, add to 1.2 ml of thioacetamide reagent, mix immediately and allow to stand for 2 minutes

Conclusion:  Any brown colour produced by test preparation should not be greater than the colour produced by standard preparation.

Limit: Not more than 0.1 ppm.

4.0  Iron

Apparatus: UV spectrophotometer

Reagent: Conc. Hydrochloric acid, Hydroxylamine HCl, O-phenanthroline

Test Preparation: To the residue saved in total dissolved solids, add 1 ml of conc. HCl, allowing the acid to flow the sides of the dish and 10 ml of distilled water. Carefully heat to boiling. Filter into a 50 ml volumetric flask, and wash the filter several times with small portion of distilled water.

Standard preparation: At the same time add to 0.3 ml, 0.5 ml, 0.8 ml and 1.0 ml of  Iron standard solution (100 mcg/ml) to separate 50 ml volumetric flask.

Colour development: Add 5 ml of Hydroxylamine HCl solution to each flask and heat to boiling, or heat in a boiling water bath for 10 minutes. Cool, and add 0.5 ml of the O-phenanthroline solution, Make the solution alkaline with ammonia. Make up the volume with distilled water. Measure the absorbance at 515 nm against reagent blank.

Plot a graph of std abs against its conc.

From the graph find out the conc. of iron in sample.

Limit: NMT 0.3 ppm

5.0  Chlorides

Apparatus: Autotitrator.

Procedure: Add 20 ml of the sample into plastic beaker and titrate against 0.05 N silver nitrate solution. Find out the chloride in terms of ppm.

Limit: Not more than 250 ppm

6.0  Sulphate

Apparatus: Balance, Muffle furnace

Reagent: Conc HCl, Barium Chloride

Procedure: Take 250 ml of the filtered sample in a 500 ml beaker and add to it 1 ml of conc. HCl. Heat to boiling till about 50 ml remains in the beaker. Add to boiling solution 10% Barium chloride solution till complete precipitation takes place. Boil gently till the precipitate settles down, allow it to stand on water bath for one hour. Filter through No.32 filter paper. Wash the residue till it free from chloride. Dry the ppt and ignite in a tared silica crucible to a constant weight.

Calculation:

                               mg of residue x 1000 x 0.608

                     = -------------------------------------------------

                                   volume of sample taken

Limit: Not more than 200 ppm

7.0  Nitrate

Reagent: Potassium chloride, Diphenylamine, Sulphuric acid

Test preparation: Place 5 ml in a test tube immersed in iced water, add 0.4 ml of a 10.0 %w/v solution of potassium chloride 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of nitrogen free Sulphuric acid. Transfer the tube to a water-bath at 50°C. Allow it to heat for 15 min.

Standard preparation: Place 4.5 ml of nitrate free water and 0.5 ml of nitrate standard solution (450 ppm NO3) in a test tube. add  0.4 ml of a 10.0 %w/v solution of potassium chloride, 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of nitrogen free sulphuric acid. Transfer the tube to a water-bath at 50°C. Allow it to heat for 15 min.

Conclusion: Any colour produced by sample should not be greater than that produced by standard preparation.

Limit: Not more than 0.2 ppm

8.0  Total Dissolved Solids

Apparatus: Balance

Procedure: Evaporate to dryness 250 ml of the filtered sample of water inconvenient portion in a tared 100 ml evaporating dish ad dry the residue at 105°C till to constant weight.

Calculation:

                                        Wt of residue in mg x 1000

                                    = -----------------------------------

                                               Volume of sample

Limit: Not more than 500 ppm

9.0  Total Hardness

Procedure:  Transfer 250 ml of the sample into a 500 ml conical flask. Add 10 ml of ammonium chloride-ammonium hydroxide buffer solution and titrate against 0.1M EDTA solution using Erichrome Black T as indicator. The end point is from wine red to a true blue colour.

Calculation:

    B.R. x actual normality  x Calcium eqv. of sol. (4.002) X 2.5 x 1000

= ------------------------------------------------------------------------------------

                       0.1 x volume of sample taken

Limit: Not more than 300 ppm

10.0  Test for total viable aerobic count & pathogens

Procedure:  For water sample suspend 1 ml of liquid in 9 ml of sterile fluid casein digest Soya lecithin (FCDSL) medium. It becomes 1:10 dilution. Pipette out 1 ml of this dilution with sterile pipette and pour into 2 sterile petri dish. Immediately add 50 x20 ml of soyabean casein digest agar medium (for TBC) and sabourads dextrose agar (for TYMC) previously melted and cool at about 45°C. Cover the petri dish and allow the contents to solidify at a room temperature Simultaneously keep the control. Invert the petri dishes and incubate SCDA agar plates at 37°C for 120 hours- (for TBC) and SDA agar plates at room temperature for 120 hours (for TYMC).

Examine the plate for growth; count the number of colonies and express in terms of microorganism per ml.

11.0  Test for Pathogens:

a) Escherichia coli:

Add 5 ml of water to 50 ml of sterile nutrient broth. Incubate at 37°C for 18 - 24 hours. This becomes enrichment culture.

Primary test: Add 1 ml of enrichment culture to the tube containing 5 ml of sterile Mac-Conkey’s broth. Incubate at 37°C for 48 hours. If the contents of the tube shows acid and gas production carry out the secondary test.

Secondary test: Add 0.1 ml of the contents of the tube showing acid and gas containing (i) 5 ml of Mac-Conkey’s broth and (ii) 5 ml of peptone water. Incubate at 37°C for 24 hours and examine tube (i) for acid and gas and tube (ii) for Indole. To test for indole, add 5 ml of Kovac’s reagent, shake well and allow to stand for one minute. If red colour is produced in the reagent layer indole is present.

The present of acid and gas and of Indole in the secondary test indicates the present the E. coli.

b) Salmonella:

Add 5 ml of water to 50 ml of sterile nutrient broth. Incubate at 37°C for 18-24 hours. This becomes enrichment culture.

Primary test: Add 1 ml of enrichment culture to each of the two tubes containing (i) 10 ml of Selenite – F broth and (ii) 10 ml of tetrathionate broth and incubate at 37°C for 48 hours.

From each of these two cultures, inoculate three plates containing a layer of (a) Brilliant green agar (b) Deoxycholate citrate agar and (c) Bismuth sulphite agar and inoculate the plates at 37° for 18-24 hours. If any of the colonies confirming to the description in the table are produced, carry on the secondary test.

Medium	Description of colony
Brilliant green agar	Small, transparent and colorless or opaque, pink or white (frequently surrounded by pink or red zone.)
Deoxycholate citrate agar	Colorless or opaque, with or without black center.
Bismuth sulphite agar	Black or green

Secondary test: Subculture any colony showing the characteristics given in the above table in triple sugar iron agar, by first inoculating the surface of the slope and then making a subculture with the same inoculating needle and at the same time inoculate urea broth. Incubate at 37°C for 18-24 hours. The formation of acid and gas in the stab culture (with or without concomitants blackening) and the absence of acidity from the surface growth in TSI, together with the absence of red colour in the urea broth, indicate the presence of salmonella. If acid but no gas is produced in the stab culture, identity of the organism should be confirmed by agglutination test.

c) Pseudomonas: 

Add 5 ml of water to 50 ml of Cetrimide broth at incubate at 37° C for 72 hours. Subculture on a plate containing a layer of Cetrimide agar and incubate at 37°C for 48 hours. Examine the resulting growth by Gram’s stain and apply the Oxidase test. Gram-negative bacilli showing a positive oxidase test indicates the presences of Pseudomonas.

Oxidase test: Place 2-3 drops of freshly prepared 1% w/v solution of NNN’ N-tetramethyl – p-phenylenediammonium dichloride on a piece of filter paper and smear with the suspected colony. If purple colour is produced within 5 – 10 seconds the test is positive.

Limit: 

(A) Total viable aerobic count: Not more than 500 CFU/ml

(B) Pathogens (Escherichia Coli, Salmonella and Pseudomonas aeruginosa: Should be absent per 100 ml.

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