ANALYSIS
OF WATER
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
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
|
|
Brilliant Green agar
|
Small, transparent, colorless or pink to white
Opaque (frequently surrounded 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
|
SOP
|
Standard Operating Procedure
|
|
M
|
Molarity
|
|
mg
|
Milligram
|
|
TSS
|
Total Suspended Solids
|
|
TDS
|
Total Dissolved Solids
|
|
ppm
|
Parts per million
|
|
TOC
|
Total Organic Carbon
|
|
%
|
Percent
|
|
°
|
Degree Centigrade
|
|
PAF
|
Pseudomonas agar for Fluorescenin
|
|
PAP
|
Pseudomonas agar for Pyocyanin
|
|
G.C
|
Giolitti Cantoni broth
|
|
TAMC
|
Total Aerobic Microbial Count
|
|
ml
|
Milliliter
|
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