SOP for Analysis of Water

Standard operating procedure to analyze the water samples for chemical and microbial tests.

1.0 PURPOSE
To lay down the procedure for the analysis of water.

2.0 SCOPE
Applicable to all sampling points of the water system.

3.0 RESPONSIBILITY
Microbiologist

4.0 ACCOUNTABILITY
Head of Department

5.0 PROCEDURE
Collect the sample as per Standard Operating Procedure for water sampling and analysis for chemical and microbiological parameters as per their
specifications.

5.1 Chemical Analysis

Prepare the solutions/ reagents for chemical analysis.

5.1.1 Description
Examine the water physically such as color, odor.

5.1.2 Hardness
Take 100 ml sample add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black 11 mixture and add of 0.01 M disodium edetate until, a pure blue
color is produced. Measures the volume of disodium edetate used and calculate the hardness by the following formula.
Hardness as mg/L = ml of EDTA used x 1000 mg/L
Sample volume

5.1.3 Total Suspended Solids (TSS)

Take the gouch crucible clean and dry in oven for one hour at 105°C, Cool the gouch crucible in desiccator and take the empty weight of gouch
crucible and then filter the 30 ml water sample from the gouch crucible with the help of vacuum pump and calculate the TSS with the help of the
formula.
TSS = W2-W1 × 1000 (mg/L)
ml of solution taken
W1 : Weight of Gouch crucible before filtration
W2 : Weight of Gouch crucible After filtration

5.1.4 Total dissolved solids (TDS)

Measure the conductivity at 25 °C with a calibrated conductivity meter and convert the value in TDS by the following formula.
TDS in mg/L= conductivity in mS X 0.667 (Geographical factor of area)

5.1.5 Acidity
Take 10 ml sample freshly boiled and cooled sample, add 0.05 ml of methyl red solution and mix, the resulting solution is not red.
Interpretation of result: If the solution is in red color the sample is Acidic

5.1.6 Alkalinity
Take 10 ml sample freshly boiled and cooled sample, add 0.1 ml of bromothymol blue solution and mix.
Interpretation of result: If the solution is in blue color the sample is Alkaline.

5.1.7 Ammonium
Take 20 ml sample add 1 ml of alkaline potassium mercuri-iodide solution and allow standing for 5 minutes. When vertically viewed the solution is not
more intensely colored than a solution prepared at the same time by adding 1 ml of alkaline potassium mercuri-iodide solution to a solution
containing 2.5 ml of dilute ammonium chloride solution and 7.5 ml of the liquid being examined.

5.1.8 Calcium & Magnesium

Take 100 ml sample add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black 11 mixture and 0.5 ml of 0.01 M disodium edetate, a pure
blue color is produced.

5.1.9 Heavy Metals

In a glass-evaporating dish evaporate 150 ml of sample to 15 ml on a water bath.
Standard solution
Into a small Nessler Cylinder, pipette 10.0 ml of lead standard solution (1ppm Pb).
Test Solution
Pipette 12 ml into a small nessler cylinder.
Procedure
To the cylinder containing the standard solution add 2.0 ml of the test solution and mix. To each cylinder add 2 ml of acetate buffer pH 3.5, mix, add
1.2 ml of thioacetamide reagent, allow to stand for 2 minutes and view downwards over a white surface, the colour produced with the test solution is
not more intense than that produced with the standard solution.

5.1.10 Chloride
Take 10 ml sample add 1 ml of 2 M nitric acid and 0.2 ml of 0.1 M silver nitrate, the appearance of the solution does not change for at least 15
minutes.

5.1.11 Nitrate
Take 5 ml sample in a test tube immersed in ice add 0.4 ml of a 10% w/v solution of Potassium chloride, 0.1 ml of diphenylamine solution and
dropwise with shaking 5 ml of sulphuric acid. Transfer the tube to a water bath at 50°C to allow standing for 15 minutes. Any blue colour in the
solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 5.5 ml of nitrate free water
and 0.5 ml of nitrate standard solution (2 ppm NO3).

5.1.12 Sulphate
Take 10 ml sample add 0.1 ml of 2 M Hydrochloric acid and 0.1 ml of barium chloride solution. The appearance of the solution does not change for at
least 1 hour.

5.1.13 Oxidisable substances

Take 100 ml sample add 10 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate and boil for 5 minutes, the solution should
remain faintly pink.

5.1.14 Residue on evaporation

Evaporate 100 ml sample to dryness into hot plate and dry to a constant weight at 105°C. The residue weighs not more than 1 mg (0.001%).
Residue on evaporation: W2-W1 × 100 (mg/L)
ml of solution taken
W1 : Weight of Evaporating dish
W2 : Weight of Evaporating dish + Residue

5.1.15 Total Organic Carbon

Analyse the sample for TOC in a calibrated TOC Analyser as per SOP.
5.1.15.1 Alert and Action limit for Total Organic Carbon of water system
S.No. Type of Water Alert Limit (ppb) Action Limit (ppb)
1 Purified water 300.0 500
2 Water for injection 250.0 500
3 Pure Steam 250.0 500

5.1.15.2 If the TOC results are above alert and action limit, follow the SOP.

5.1.16 Conductivity
Take the 100 ml sample in a suitable container, and stir the test sample by maintaining the temperature 25°C ± 1°C, measure the conductivity with
the help of calibrated conductivity meter.
Temperature and the respective Conductivity.
Temperature (°C) Conductivity µS cm‾ 1
0 0.6
5 0.8
10 0.9
15 1.0
20 1.1
25 1.3
30 1.4
35 1.5
40 1.7
45 1.8
50 1.9
55 2.1
60 2.2
65 2.4
70 2.5
75 2.7
 80 2.7
85 2.7
90 2.7
95 2.9
100 3.1

5.1.17 pH
Take 100 ml of sample and add 0.3 ml of saturated KCL solution. Mix the solution well and then measure the pH with the help of Calibrated pH
meter.
NOTE : If results are observed out of limit in chemical analysis of water, follow the SOP.

5.2 Microbiological Analysis

Analyse the water samples for Microbiological analysis as per specifications.

5.2.1 Pour Plate Method

Dispense one ml of sample into two Petri dishes. Approximately add 15-20 ml of R2A / Plate count Agar into each Petri dishes. Cool the media
approximately 45°C (feel on the dorsal side of the hand, it should be bearable). Cover the Petri dish, mix the sample with the agar by tilting or rotating
the dishes and allow the contents to solidify at room temperature. Invert the Petri dishes and incubate at 30-35°C for 5 days. After incubation,
examine the plates for growth, count the number of colonies and express the average for the two plates in terms of the number of colony forming
units per ml.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria

5.2.2 Membrane Filtration Technique

The procedure gives the use of a single disposable/ autoclaveable filtration funnels and filter holder, using MILLIFLEX system.
Preparation of the Filtration apparatus
Operate the Milliflex as per its SOP. Use sample size as specified in the specification for filtration through the 0.45 m filter.
After completion of filtration of the sample, rinse the filter with 100 ml sterile water remove the filter using sterilized forceps and transfer it immediately
to the previously prepared petri-dish with appropriate medium (R2A agar/Plate count agar).
Place the membrane filter carefully so that the air should not be trapped inside the filter, as this will prevent the nutrient medium from reaching the
entire membrane surface. Replace the lid. Incubate the plates in an upright position (in case of filter) at 30-35°C for 5 days. Count the number of
colonies on the membrane and express the results as per specification.

5.2.3 Bacterial Endotoxin Test

Refer the SOP for bacterial endotoxin test.

5.2.4 Pathogens
The sample shall be tested for the following four specific pathogens.
(A) Salmonella species
(B) Escherichia coli
(C) Pseudomonas aeruginosa
(D) Staphylococcus aureus
Filter 100 ml of water sample through the 0.45 membrane filter fixed on Milliflex system. After filtration removes the filter aseptically and put it in 100
ml Soybean Casein Digest Medium and incubate at 30-35°C for 24-48 hours.
From Soybean Casein Digest Medium, inoculate sterile 10 ml volumes of the following enrichment broths using 1 ml of inoculated broth
1. Selenite Cystine Broth for Salmonella species.
2. Tetrathinate Broth for Salmonella species.
3. MacConkey’s Broth for Escherichia coli
4. Cetrimide Broth for Pseudomonas aeruginosa
5. Giolitti Cantoni Broth for Staphylococcus aureus (use sterile liquid paraffin for anaerobic conditions).
Incubate the tubes for 24-48 hours at 30-35°C.
(A) Test for Salmonella species:
If growth is present in Selenite Cystine Broth and Tetrathionate Broth, inoculate the following selective media plates and incubate at 30-35° C for 24-
48 hours for presumptive identification of the pathogen.
Medium Description of Colony
Xylose-Lysine Deoxycholate agar medium Red with or without Black Centre
Bismuth Sulphite agar medium Black or Green colonies
Small, transparent, colorless or pink to white Opaque (frequently surrounded
Brilliant Green agar
by pink to red zone)

Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests with the following biochemical/media and by gram
reaction.
Individually transfer the suspected colony by first streaking the slope of slant, of Triple Sugar-Iron Agar with inoculating loop and then stabbing with
inoculating straight wire well in the butt.
Incubate at 30-35° C for 24-48 hours
After incubation, examine the tube of Triple Sugar Iron Agar Medium for the presence of microbial growth and for the following Physical
characteristics.
(a) Slant Surface: Alkaline reaction (red color)
(b) Butt: Acid reaction (yellow color) and/or gas bubble (with or without concomitant blackening).
If the butt, slant of Triple Sugar Iron Agar shows growth and physical characteristics conforming to the above descriptions the presence of Salmonella
species is indicated.
(B) Test for Escherichia coli
If the inoculated MacConkey’s broth tube shows acid and gas formation, inoculate the following selective media plates and incubate at 30-35°C for
24-48 hours for presumptive identification of the pathogen.
Medium Description of Colony
MacConkey’s Agar Brick red may have surrounding zone of precipitated bile.
Eosin Methylene Blue Agar Metallic sheen with dark grey colonies

Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests into the following bio-chemicals/media and by gram
reaction.
Add 0.1 ml of the contents of the tube showing acid and gas to tubes containing 10 ml of peptone water
From peptone, water tube perform Indole test as follow
Add 0.5 ml of Kovac’s reagent to peptone water tube, allow to stand for one minute, if a red color is produced in the reagent layer indole is present
The presence of acid and gas in MacConkey's broth, in peptone water and indole, indicates the presence of Escherichia coli.
Presence of Escherichia coli shall be confirmed by Gram staining (Gram-ve rods) and by streaking a loopful of the MacConkey’s broth, with acid and
gas production on plates of MacConkey Agar, and Levine Eosin Methylene Blue Agar.
Incubate the plates at 30-35°C for 24-48 hours.
If after incubation, plates show colonies of following characteristics presence of Escherichia coli is confirmed.
MacConkey’s Agar: Brick red colonies with or without surrounding zone of precipitates.
Levine Eosin Methylene blue Agar: Colonies with characteristic of metallic sheen under reflected light and blue-black appearance under transmitted
light.
(C) Test for Pseudomonas aeruginosa
If the inoculated Cetrimide broth tube shows growth with greenish/bluish pigmentation, inoculate the following selective media plates and incubate at
30-35°C for 24-28 hours for presumptive identification of the pathogens.
Medium Description of Colony
Cetrimide Agar Greenish colonies, which exhibit a greenish fluorescence under ultra violet light.
Pseudomonas Agar (For Pyocyanin) Colourless to yellowish, yellowish under ultra violet light.
Pseudomonas Agar (For Fluorescein) Colourless to yellowish, yellowish under ultra violet light.

Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests
Streak suspected colony on Pseudomonas Agar for Fluorescenin (PAF) Detection and Psedomonas Agar for Pyocyanin (PAP) Detection using
inoculating loop. Incubate the plates in inverted condition at 30-35°C for 24-28 hours. Simultaneously inoculate the suspected colony in 100 ml of
Soyabean casein digest medium and incubate at 41° to 43°C for 18 to 24 hours.
After incubation, examine the plates and tube of Soybean casein digest medium for the presence of microbial colonies of Gram-Negative rods
exhibiting following characteristics. Pseudomonas Agar for fluorescenin detection: Colorless to yellowish fluorescence under the ultra violet light.
Pseudomonas Agar for Pyocyanin Detection: Greenish colonies, which exhibit a blue fluorescence ultraviolet light. Soybean casein digest medium:
Growth occurs.
If colonies are found confirming to above descriptions, Oxidase test shall be performed to confirm identification as follow:
With the aid of an inoculating loop, transfer suspected colonies to strip or discs of filter paper impregnated with N, N-dimethyl-p-
phenylenediamine dihydrochloride.
If a Pink-Purple colour is produced within five to ten seconds, the presence of Pseudomonas aeruginosa is confirmed.
(D) Test for Staphylococcus aureus
If growth is present in Giolitti Cantoni (G.C) broth, usually characterized by black settled growth at the bottom of the broth under anaerobic
conditions, inoculate the following selective media plates and incubate at 30-35°C for 24-28 hours for presumptive identification of pathogen.
Medium Description of Colony
Mannitol Salt Agar Medium Yellow colonies with yellow zones
Vogel Johnson Agar Medium Black surrounded by yellow zone
Baird Parker Agar Medium Black, shiny, surrounded by clear zones of 2-5 mm

Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests
If colonies are found confirming to the above descriptions identification shall be performed by a coagulase test as follow.
With the aid of an inoculating loop, individually transfer suspected colonies to separate tubes containing 0.5 ml of mammalian plasma (preferably
rabbit or horse).
Incubate in a water-bath / incubator at 370C for 3 to 24 hours, in parallel with positive control using known strain of Staphylococcus aureus and
negative control using Plasma alone.
Examine after 3 hours and at suitable intervals thereafter for the presence of coagulation.
If coagulation in any degree is observed, the presence of Staphylococcus aureus is indicated. And perform the gram staining for the presence of
gram Positive cocci.

5.2.5 Coli forms

Filter 100 ml of test sample and transfer the filter to M-Endo agar and incubate at 35°C for 22-24 hrs count colonies that are pink to dark red with a
green metallic surface sheen, the sheen may vary from pinpoint to complete coverage of colony. Report the as number of Coliforms colonies per 100
ml.
5.2.6 After completion of testing prepare a test report.
5.2.7 If the counts obtained are above the limits specified below investigate the results and take necessary actions as per SOP.

5.3 Alert and Action limit for TAMC of water system

S.No Type of water Alert limit Action limit
1 Raw water 300 500
2 Soft water 200 500
3 Potable water 150 500
4 Drinking water 100 500
5 Purified water 50 100
6 Water for injection (100 ml) 3 10
7 Pure steam (100 ml) 3 10

If the Microbial results are above alert and action limit, follow the SOP for Out of Specification.

6.0 ABBREVIATIONS
6.1 SOP - Standard Operating Procedure
6.2 M - Molarity
6.3 mg - Milligram
6.4 TSS - Total Suspended Solids
6.5 TDS - Total Dissolved Solids
6.6 ppm - Parts per million
6.7 TOC - Total Organic Carbon
6.8 % - Percent
6.9 ° - Degree Centigrade
6.10 PAF - Pseudomonas agar for Fluorescenin
6.11 PAP - Pseudomonas agar for Pyocyanin
6.12 G.C - Giolitti Cantoni broth
6.13 TAMC - Total Aerobic Microbial Count
6.14 ml - Milliliter