Water Analysis Methods

Physical Analysis
Boiling Point (oC)
pH
Electrical Conductivity (µS/cm)
Total Dissolved Solids (mgL-1 / ppm)
Total Suspended Solids (mgL-1 / ppm)

Chemical Analysis
Total Water Harness (mgL-1 / ppm)
Determination of Calcium (mgL-1 / ppm)
Determination of Magnesium (mgL-1 / ppm)
Chlorine (mgL-1 / ppm)
Sulphate (mgL-1 / ppm)
Nitrate (mgL-1 / ppm)
Sodium (mgL-1 / ppm)
Potassium (mgL-1 / ppm)
Total Acidity (mgL-1 / ppm)
Total Alkalinity (mgL-1 / ppm)

Microbiological Testing
Heterotrophic Plate Count (CFU/mL)
Colliform Bacteria (CFU/mL)
E Colli Bacteria (CFU/mL)

Reagents and Solutions

All reagents must be of analytical grade. Use clean glassware by soaking in detergent for 24 h
and in 10% hydrochloric acid or nitric acid. Then rinse thrice with bi-distilled water and
deionized water. Use double distilled ware or deionized water for solutions preparation.

Physical Analysis of Water Samples

Estimation of Boiling Point (oC)

Boiling point of water sample can be determined by using thermometer or boiling points of
samples determine by thermometer in ignition tube. Put water sample into ignition tube and then
place in beaker full of paraffin oil (BP is above from water), to keep temperature uniform stirrer
till boiling point of sample in ignition tube achieve then note reading of thermometer carefully.
Estimation of pH
Almost all processes containing water have a need for pH measurement. The pH of water can be
measured by using pH meter. Prior to measurement of pH, first calibrate pH meter with standard
buffers of pH 4.00, 7.00 and 9.00 (Merck).

Type of Water pH Range

Soft Water 5.3 – 7.4
Hard Water 7.6 – 8.8
Sea Water 8.2 – 9.2
Water Affected by Acidic Pollutants 2.2 – 4.8
pH of Water in Equilibrium with Atmosphere 5.6

Estimation of Electrical Conductivity (µS/cm)

The conductivity of precipitation samples depends on the concentrations of the various ion
species and their different ability to transport electric charges in a solution, i.e. the ion species
equivalent conductivity. This conductivity is temperature dependent and increases approximately
2% per degree in aqueous solutions for most ion species.

Chemicals
1. Deionized water, conductivity < 0.5 µS/cm
2. Potassium chloride

Calibration
Calibration of the conductivity instrument and conductivity cell can be done by the help of
solution of a known value of conductivity or calibration standard solution. For this purpose we
use the solution of KCl of different known concentrations to determine the cell constant of the
conductivity cell and then determine the calibration frequency of cell.

Calibration Solutions

Solution Concentration Conductivity Upper Limit Lower Limit

M KCl µS/cm µS/cm µS/cm
A 0.0500 6668 6801 6535
B 0.0200 2767 2822 2711
C 0.0100 1413 1441 1395
D 0.0050 717.8 735 700
E 0.0010 147.0 149 145
F 0.0005 73.9 77.8 70.2
G 0.0001 14.94 16.5 13.5
Measurement of Conductance of Water

1. Calibrate the probe (glass electrode) using a standard solution of known conductivity and
check calibration by measuring the EC of the standard solutions.
2. Collect sample water in a glass or plastic container. Take enough water sample in a
beaker so the electrode tip submerge in sample; rinse the electrode with deionize water or
with sample and dry with soft tissue before inserting the electrode into the sample vessel /
beaker.
3. Submerge the pH electrode into the sample and wait until the EC reading on the meter
stabilizes and then adjust a knob on the meter to correct the EC for temperature. Record
the readings when the EC reading is stable.

Total Dissolved Solids (TDS)

Dissolved solids can affect the color or test of water and, if very high, may be an indication that
the water sample is saline. Determination of total dissolved solids can be determined by
evaporating a known volume of the filtered water sample. The increase in mass of the container
is then determined.

Procedure
Wash, dry and weigh the beaker. Pipette 50 mL of water sample (precisely note volume / for
precise volume measurement use pipette or burette) and filter through filter paper. Then dry
filtrate of beaker in oven after evaporating the filtrate cool the beaker and then note down weight
of it. Repeat this step until to get constant weight of beaker with residue in it.

Total Dissolved Solids (mg/L or ppm) =

A= Weight of dry residue + beaker (mg)

B = Weight of beaker (mg)

Total Suspended Solids (TSS)

The main physical problem that may be cause by suspended solids in natural water bodies is that
they cut down light transmission through the water and so lower the rate of photosynthesis in
plants. Also in less turbulent parts of the river some of the solids may sediment out, smothering
the life on the river bed. The suspended solid determination is extremely valuable in the analysis
of polluted waters. It is one of the major parameters used to evaluate the strength of domestic
waste waters and to determine the efficiency of treatment units. The analysis of the suspended
solids can be accomplished by filtration and weighing once again.

Procedure
Filter 50 mL of water sample (for precise volume measurement use pipette or burette) through
weighed filter paper and then dry filter paper in oven, after cooling note weight of residue with
filter paper and then subtract weight of the filter paper. Repeat this step until to get constant
weight of filter paper with residue on it.

Total Suspended Solids (mg/L or ppm) =

A= Weight of dry residue + filter paper (mg)

B = Weight of filter paper (mg)

Chemical Analysis

Total Water Hardness

The hardness of water is generally due to dissolved calcium and magnesium salts and may be
determined by complexometric titration. Ethylenediaminetetraacetic acid and its sodium salts
(abbreviated EDTA) form a chelated soluble complex when added to a solution of certain metal
cations. If a small amount of a dye such as Eriochrome Black T or Calmagite is added to an
aqueous solution containing calcium and magnesium ions at a pH of 10.0 ± 0.1, the solution
becomes wine red. If EDTA is added as a titrant, the calcium and magnesium will be complexed
with EDTA and when all of the magnesium and calcium has been complexed the solution turns
from wine red to blue, marking the end point of the titration.

Reagents

Buffer Solution: (ammonium hydroxide – ammonium chloride)

Add 70 mL of concentrated ammonia solution to 8g ammonium chloride and dilute to 250 mL
with deionized water.

Eriochrome Black – T (EBT) Indicator

Add 2 g Eriochrome Black – T in 50 mL ethanol.

Na2EDTA Solution
Prepare 0.01 M disodium salt of ethylenediamine tetra acetate (C10H14N2Na2O8 .2H2O, MW =
372.24 g/mole) in deionized water. Concentration of EDTA solution will decide as per
requirement.

Standard Magnesium Sulphate (MgSO4 .7H2O) Solution

Prepare 0.01M standard solution of magnesium sulphate (MW = 246.48 g/mole) in deionized
water and make volume up to the mark of volumetric flask.
Standardization of EDTA Solution
Standardize 0.01 M EDTA solution by titrating it with standard 0.01M magnesium sulphate
solution at pH 10 using 2-3 drops of Eriochrome Black-T (EBT) indicator. Also run the blank
(deionized water or distilled water used for solutions preparation) by following same procedure.
End point of titration is blue from red. From concordant reading of titration calculate
concentration of EDTA.

Procedure
In 25 mL water sample (accurately measure) add 10 mL of buffer solution of pH 10 (ammonium
hydroxide – ammonium chloride) or pH of solution should be 10 then add 2-3 drops Eriochrome
Black-T (EBT) indicator then titrate with standard EDTA solution until the color change from
red to pure blue. The total hardness is expressed in parts of CaCO3 per million of water.

Total Water Hardness in mg/L or ppm as CaCO3 =

= Volume of titrant (standard EDTA) consumed for sample in mL

= Volume of titrant (standard EDTA) consumed for blank in mL
= Molarity of standard EDTA solution
= Volume of sample in mL
100000 = Conversion factor
Determination of Calcium

Generally calcium is analyzed by titration method. Other high tech method involves equipment
like auto-titrator and ion chromatographs, atomic absorption spectrophotometric method and
inductively coupled plasma method.

When add standard EDTA solution (after standardization of EDTA) to water containing both
calcium and magnesium, EDTA combines first with calcium. When sodium hydroxide buffer is
added to water magnesium is precipitated as magnesium hydroxide and EDTA combines with
calcium only, a color change is observed when all the calcium is titrated with standard EDTA at
a pH of 12-13.

Procedure
Take 25 mL water sample in a conical flask and add I M NaOH to maintain the pH 12-13 of the
sample then add pinch of Calcon or Murexide (ammonium purpurate) indicator. Shake it till the
indicator get dissolved and pink / red color is produced. Then titrate against the standard EDTA
solution; add EDTA slowly with constant stirring till color change from pink to pure blue in case
of using Calcon and purple color (end point) if using Mureoxide.

Number of replicates should be “Three” for each sample.

Calcium in Water Sample in mg/L or ppm =

= Volume of titrant (standard EDTA) consumed for sample in mL

= Volume of titrant (standard EDTA) consumed for blank in mL
= Molarity of Standard EDTA solution
= Volume of sample in mL
1000 = Conversion factor
40 = Atomic weight of Calcium (Conversion factor)

Determination of Magnesium

Magnesium is present in seawater in amount of about 1300 ppm. After sodium it is the most
commonly found cation in oceans. Rivers contain approximately 4 ppm of magnesium marine
algae 6000 – 20000 ppm and oysters 1200 ppm. The human body contains about 25 g of
magnesium, of which 60% in the bones and 40% is present in muscle and tissues. It is a dietary
mineral for humans. Magnesium is an ingredient of many enzymes.

Evaluating Magnesium by Calculation Method

Once calcium and total hardness is analyzed by EDTA titrimetric method, their results are used
in calculation of magnesium in same sample by using formula written below.
Calculate magnesium by using the formula:

Mg in mg/L or ppm = Total Hardness of Water as mg CaCO3 - Calcium Hardness (as CaCO3 / L) o.243

Where calcium hardness is calculated by multiplying Ca with 2.5

Acidity
Acidity of water is its quantitative capacity to react with a strong base to a designated pH. The
rates of chemical reactions, biological processes and corrosively are all influenced by acidity of
the water sample. Hydrogen ions present in a sample as a result of dissociation or hydrolysis of
solutes react with additions of standard alkali. Acidity thus depends on the end-point pH or
indicator used.

Measurement of Acidity
The acidity of water samples can be determined by titration of water with standard NaOH in the
presence of phenolphthalein as an indicator. The color change from colorless to pink as an end
point of the reaction.

Reagents

Preparation of 0.1M NaOH Solution

Weigh amount of NaOH for preparation of 0.1M solution of it then transfer weighed amount into
a 250 mL volumetric flask and make up volume with distilled or deionized water up to the mark.

Preparation of Standard 0.05M Oxalic acid (C2H2O4. 2H2O) Solution

Prepare 0.05M oxalic acid solution in a 100 mL volumetric flask and fill the flask with distilled
or deionized water.

Procedure
Standardization of NaOH
Standardize 0.1M NaOH solution with 0.05M standard oxalic acid (C2H2O4.2H2O) solution by
using Phenolphthalein as an indicator.
After standardization of NaOH, if require dilute it as per requirement for water sample analysis.
In 50 mL of water sample add 2 to 3 drops of Phenolphthalein and titrate with standard solution
of NaOH color change from colorless to pinkish as an end point.
 Acidity in mg/L or ppm =

= Volume of titrant (standard NaOH) consumed for sample in mL

= Volume of titrant (standard NaOH) consumed for blank in mL
= Molarity of standard NaOH solution
= Volume of sample in mL
50000 = Conversion factor

Alkalinity

To determine the alkalinity, titrate a known volume of water sample with a standard solution of
strong acid to a pH value of approximately 4 or 5. Titrations can be used to distinguish between
three types of alkalinity: hydroxide, carbonate, and bicarbonate alkalinity. Carbonate alkalinity is
determined by titration of the water sample to the phenolphthalein or metacresol purple indicator
endpoint at approximately pH 8.3. Total alkalinity is determined by titration of the water sample
to the endpoint of the methyl orange. The difference between the two is the bicarbonate
alkalinity. Hydroxide (OH-) alkalinity is present if the carbonate or phenolphthalein alkalinity is
more than half of the total alkalinity [American Water Works Association (AWWA), 1992].

Reagents

0.1M H2SO4 Solution or 0.1M HCl Solution

Prepare 0.1M of H2SO4 (18M, 36N) or 0.1M HCl (9.8 M, 9.8N) solution in 250 mL volumetric
flask and make up the volume with distilled or deionized water.

Standard 0.05M Sodium carbonate (Na2CO3) Solution

Take sufficient quantity of standard Na2CO3 salt in a watch glass or small beaker and dry at 250
o
C for 4 hr in oven to remove traces of moisture if present and after heating cool in desiccators
and then take accurate amount of standard Na2CO3 as per requirement to prepare 0.05M solution
of it in 100 mL volumetric flask and fill the flask with distilled or deionized water.

Methyl Orange Indicator Solution

Dissolve 0.125g methyl orange in distilled or deionized water and make volume up to the mark
in 100 mL.

Procedure

Standardization of H2SO4 (0.1M) or 0.1M HCl Solution

Standardize 0.1M H2SO4 or 0.1M HCl Solution with 0.05M standard sodium carbonate solution
by using methyl orange as an indicator. Color change from yellow to pink as an end point.

Total Alkalinity: (Methyl Orange Alkalinity) Measurement

Total alkalinity is measured by titrating a measured volume of a water sample against a standard
acid solution in presence of methyl orange indicator. In 25 mL of water sample, add 2 to 3 drops
of methyl orange and titrate with standard solution of H2SO4 or HCl, the color change from
yellow to pinkish as an end point.

Total Alkalinity in mg/L or ppm =

= Volume of titrant (standard H2SO4 or HCl solution) consumed for sample in mL

= Volume of titrant (standard H2SO4 or HCl solution) consumed for blank in mL
= Molarity of standard H2SO4 or HCl solution
= Volume of sample in mL
100000 = Conversion factor
 Existence of Hydroxide, Bicarbonate and Carbonate at different pH

Carbonate Alkalinity (Phenolphthalein Alkalinity)

Carbonate alkalinity is determined by titration of the water sample to the phenolphthalein (end
point pink to colorless) or metacresol purple indicator. To determine this alkalinity, a known
volume of water sample is titrated with a standard solution of strong acid (H2SO4 solution or HCl
solution).

Carbonate Alkalinity in mg/L or ppm =

= Volume of titrant (standard H2SO4 or HCl solution) consumed for sample in mL

= Volume of titrant (standard H2SO4 or HCl solution) consumed for blank in mL
= Molarity of standard H2SO4 or HCl solution
= Volume of sample in mL
100000 = Conversion factor

Inter Conversion of Carbonates and Bicarbonates

Carbonic acid is formed when carbon dioxide gas is dissolved in water.

H2O + CO2 --------- H2CO3 these are the salts of carbonic acid.

These anions are formed from carbonic acid by removing H+ ions successively as follows:

H2CO3 ----------- HCO - + H+ ----------- CO3-2+H+

Bicarbonate Alkalinity
The difference between the two (Total Alkalinity and Carbonate Alkalinity) is the bicarbonate
alkalinity.
Bicarbonate Alkalinity = Total Alkalinity - Carbonate Alkalinity

Hydroxide Alkalinity
Hydroxide (OH-) alkalinity is present if the carbonate or phenolphthalein alkalinity is more than
half of the total alkalinity [American Water Works Association (AWWA), 1992]. Thus, the
hydroxide alkalinity can be calculated as two times the carbonate or phenolphthalein alkalinity
minus the total alkalinity.

Hydroxide Alkalinity = 2×Carbonate Alkalinity - Total Alkalinity

Determination of Chloride by Argentometric Method

This method is applied for drinking water. In a neutral or slightly alkaline solution, potassium
chromate can indicate the end point of the silver nitrate titration of chloride. Silver chloride is
precipitated quantitatively before red silver chromate is formed.

Equation representing overall reaction

AgNO3 + NaCl  AgCl + NaNO3 Eq (1)

2Ag+ + CrO4-2  Ag2CrO4 (red silver chromate ) Eq (2)

Reagent
Potassium Chromate Indicator Solution
Prepare indicator solution by dissolving 25g K2CrO4 or 0.02M in 50 mL distilled or deionized
water.

0.01M AgNO3 (Silver Nitrate)

Take a measured amount of AgNO3 for the preparation of 0.01M solution and dissolve in 250
mL volumetric flask and make up the volume with distilled or deionized water.

0.01M Standard NaCl Solution

Weigh accurate amount of NaCl to prepare 0.01M solution in 100 mL volumetric flask and make
volume with distilled or deionized water.
Procedure

Standardization of 0.01M AgNO3 (Silver Nitrate)

Take AgNO3 solution in the burette and accurately pipette out standard NaCl (10 mL) solution in
the conical flask and add 1 mL K2CrO4 (use as an indicator). Add silver nitrate from the burette
into the conical flask till the end point achieve (red silver chromate is formed). Calculate the
concentration of AgNO3 solution used for water samples analysis for determination of chloride.

Titration for a Blank (Distilled or Deionized Water)

In 25 mL distilled or deionized water, add 1 mL K2CrO4 (use as an indicator) and titrate with
standard AgNO3 solution to a red end point.

Titration for Water Sample

Take 25 mL water sample in 250 mL Erlenmeyer flask. Add 1 mL K2CrO4 (use as an indicator)
and titrate with standard AgNO3 solution to a red end point.

Concentration of Cl- in mg/L or ppm =

V = Volume of sample in mL
VA =Volume of standard AgNO3 used for sample in mL
VB = Volume of standard AgNO3 used for blank in mL
M= Morality of standard AgNO3
35.5 = Conversion factor

Determination of Sulfate

Sulphate ions (SO42-) react with barium ions (barium chloride BaCl2) in hydrochloric acid (HCl)
medium to form slightly soluble barium sulphate (BaSO4). The resulting turbidity is determined
photometrically at 420 nm.

Apparatus
Spectrophotometer, for use at 420 nm with matched silica cells of 1-cm or longer light path.

Reagents

Barium Chloride Crystals (BaCl2)

Add 0.05g of BaCl2 in each standard solution and in water samples.
Conditioning Reagent
To prepare conditioning reagent take 15g NaCl and dissolve in 60 mL of deionized water and
then add 6 mL HCl, 10 mL glycerol and 20 mL of 95% ethanol.

Stock Solution of Sulfate

Prepare 1000 ppm stock solution of sulfate from Na2SO4 or K2SO4 Analytical grade salts in 100
mL distilled or deionized water. From this stock solution prepare required diluted solution.

Procedure

Spectrophotometric Measurement
Prepare dilute standard solutions of sulphate in the range of 10 to 100 ppm or as per requirement
from 1000 ppm stock solution and then record the absorbance of each standard solution, water
samples and make a calibration curve. Transfer accurately measured solutions of each standard
(25 mL) and water samples (25 mL) into individual Erlenmeyer flasks and add 1.25 mL of
conditioning reagent with constant stirring by magnetic stirrer. Then add barium chloride crystals
(0.05g) and start stopwatch. Record the absorbance on spectrophotometer after each 30 seconds
up to 4 minutes at 420nm. Select the maximum absorbance from interval of 2 minute. Repeat this
with all standards and water samples. Also run a blank (25 mL distilled or deionized water)
following same procedure. After record of absorbance for blank minus this absorbance from all
standards and water samples absorbance to get corrected absorbance to use for calibration curve.
The sulfate concentration in water samples can be calculated from calibration curve.

Calibration Curve for Sufate

0.7
Absorbance

0.6
0.5
0.4 y = 0.0062x
0.3
R² = 0.9973
0.2
0.1
0
0 20 40 60 80 100 120
Concentartion (ppm)
Determination of Nitrate

Measurement of UV absorption at 220 nm enables rapid determination of NO3–. Because

dissolved organic matter also may absorb at 220 nm and NO3– does not absorb at 275 nm, a
second measurement made at 275 nm may be used to correct the NO3– value.

Apparatus
Spectrophotometer use at 220 nm and 275 nm with matched silica cells of 1-cm or longer light
path.

Reagent

Nitrate-free Water
Use deionized water of highest purity for preparation of all solutions.

Stock Solution of Nitrate

Prepare 1000 ppm stock solution of nitrate from analytical grade NaNO3 or KNO3 salts in 100
mL deionized water.
Intermediate Nitrate Solutions
Prepare dilute standard solutions of nitrate in the range of 10 to 100 ppm or as per requirement
from 1000 ppm stock solution.

Procedure
Record the absorbance of each standard solution at 220 nm and 275 nm. For standards and
samples, subtract two times the absorbance reading at 275 nm from the reading at 220 nm to
obtain absorbance due to NO3–. Construct a standard curve by plotting absorbance due to NO3–
against standard concentration. Using corrected sample absorbance, obtain sample
concentrations directly from calibration curve.

Calibration curve for Nitrate

0.2
y = 0.0558x
0.15
Absorbance

R² = 0.9985
0.1

0.05

0
0 0.5 1 1.5 2 2.5 3 3.5
Concentration (ppm)
Determination of Sodium and Potassium

Preparation of Samples
Water samples can be cleared of organic matter by wet ashing method. Evaporate Two hundred-
mL sample of water to 20 mL on a hot plate and add 5 mL HNO3; keep continue heating until the
solution clear. Then cool it to room temperature before filter it to remove insolubles and make
the volume up to mark in100 mL volumetric flask with deionzed water.
Before analyzing samples run series of standard solutions on Atomic Absorption
Spectrophotometer or Flame Photometer. From calibration curve calculate concentration of
sodium and potassium in samples.

Micro-Biological Testing

Heterotrophic Plate Count (CFU/mL)

Total aerobic microbial count in water:
Material
1. Cover water bath, with circulating system to maintain temperature of 45.5 ± 0.2°C.
2. Incubator, 35 ± 1.0°C.
3. Sterile graduated pipettes, 1.0 and 10.0 mL.
4. Dilution bottles.
5. Plate count agar 3.

Procedure for Preparation

1. Weigh 12.6g nutrient agar.
2. With gentle stirring on a hotplate-stirrer, slowly add the agar to 450 mL of cold
demineralized or deionized water in an 800 mL beaker.
3. When addition of the agar complete then heats the mixture with vigorous stirring and boil
for 15 minutes.
4. Remove the beaker from the stirrer hotplate, retrieve the stirrer “flea” using a magnet,
and cover the beaker (foil).
5. Allow to cool to 50°C.
6. Store in the refrigerator (short term only).

Preparation of dilutions of sample

1. Take three bottles or test tubes A, B and C in each of which fill 9 mL of demineralize
water and mark with 1/10, 1/100, and 1/1000 respectively.
2. 1/10 dilution is form when1mL of sample transfer to the bottle A.
3. 1/100 dilution is form when 1mL sample from A is transfer into bottle B.
4. 1/1000 dilution is form by transporting 1 mL from bottled B to C.
Pour Plate Technique

The Pour Plate technique can be used on any type of liquefied sample for the enumeration of
bacteria. Conditions vary depending upon the type(s) of bacteria being enumerate.
Serial dilutions of 1/10, 1/100, 1/1000 will use for incubation, one of the dilutions will yield
growth of 30-300 colonies (the ideal counting range) on the agar plate.
Transfer 1.0 mL of the sample or dilution to a sterile, empty Petri dish by heating in boiling
water, and then allow to cool in a water bath to 44-46°C.
Pour approximately 15 mL of agar medium into the Petri dish containing the sample. Mix sample
and agar thoroughly by rotating the plate several times, clockwise, then counterclockwise.
When the media solidify, invert the plates and incubate 48 to72 hours at 30oC to 35oC.
Following the appropriate length of incubation, the colonies will count and determine the
CFU/mL.

Calculation
CFU/mL = Number of Colonies × Dilution Factor

Colliforms (CFU/mL)
Material
1. Incubator, 35 ± 1.0°C.
2. Sterile graduated pipettes, 1.0 and 10.0 mL.
3. Dilution bottles.
4. EMB ager.
5. Preparation of Dilutions of Sample.
6. Three bottles A, B and C each of which fill with 9 mL of demineralize water and mark
with 1/10, 1/100, and 1/1000 respectively.
7. 1/10 dilution is form when1mL of sample transfer to the bottle A.
8. 1/100 dilution is form when 1mL sample from A is transfer into bottle B.
9. 1/1000 dilution is form by transporting 1 mL from bottled B to C.

Preparation of EMB Medium

For media preparation follow the manufacturer instructions:
1. Do weight 36 grams of medium.
2. With gentle stirring on a hotplate-stirrer, slowly add the agar to 1000 mL or 1L of cold
demineralized water.
3. When addition of the agar is complete, heat the mixture with vigorous stirring and boil
for 15 minutes.
4. Sterilize in autoclave at 121°C.
5. Allow to cool to 50°C.
6. Store prepared media under refrigeration (generally not to exceed 4 weeks).
Determination of Coliform

The Pour Plate technique is also used for determination of Coliform and E.coli quantitatively.
1. Serial dilutions of 1/10, 1/100, 1/1000 of water sample will yield growth of 30-300
colonies (the ideal counting range) on the agar plate.
2. Transfer 1 mL of the sample or dilution to a sterile, empty Petri dish then heat in boiling
water, and allow to cool in a water bath to 44-46°C.
3. Approximately 15 mL of agar medium pour into the Petri dish containing the sample.
Then mix sample and agar thoroughly by rotating the plate several times, clockwise, then
counterclockwise.
4. When the media is solidify, invert the plates and incubate 48 to72 hours at 30°C to 35°C.
5. After incubation the different colonies of coliform will produce on the medium. The
E.coli will differentiate by producing a specific colonies having green metallic sheen.

Calculation
E.Coli (CFU/mL) = Number of Colonies × Dilution Factor