Microbiology of Potable Water
• Direct detection of pathogenic bacteria and
viruses and cysts of protozoan parasites
requires
-- costly
-- Time-consuming procedure
-- Well-trained labor
• These requirements led to the concept of
indicator organisms of fecal pollution.
• 1914, the U.S. public health service adopted
the coliform group as an indicator of fecal
contamination of drinking water
Criteria for an ideal indicator organism:
• A member of intestinal microflora of warm
blooded animals
• Should be present when pathogens are present
and absent in uncontaminated samples
• Should be present in greater numbers than the
pathogens
• Should be at least equally resistant as the
pathogens to environmental factors and
disinfection in water and wastewater treatments
plants
• Should not multiply in the environment
• Should be detectable by means of easy, rapid,
and inexpensive methods
• Should be non-pathogenic
Proposed or commonly used microbial
indicators are the following:
Total Coliforms:
• The total group of coliforms includes the aerobic
and facultative anaerobic, gram-negative, non-
spore-forming, and rod-shaped bacteria
• Ferment lactose with gas production within 48 hr
at 35oC
• Includes E. coli, Enterobacter, Klebsiella and
Citrobacter
• 2x109 coliforms per day per capita discharges
in human and animal feces
• Not all of them are fecal in origin
• These indicators are useful for determining the
quality of potable water, shellfish harvesting
waters, and recreational waters.
• Less sensitive than viruses or protozoan cysts
to environmental factors and disinfection
• Klebsiella sometimes grow in industrial and
agricultural waste water.
• Total coliforms are one of the best indicator of
the treatment efficiency of water treatment
plants.
Fecal Coliforms:
• Include all coliforms that can ferment at
44.5oC
• Comprises bacteria such as E. coli and K.
pneumoniae
• Fecal coliforms indicates the presence of fecal
material from warm blooded animals.
• Cannot be differentiated between human and
animal contaminants
Fecal Streptococci:
• Comprises of S faecalis, S bovis, S equinus and S.
avium
• Commonly inhabit the intestinal tract of humans
and warm blooded
• Used to detect fecal contamination
• Persist well but do not reproduce in the
environment
• Fecal streptococci group, the enterococci (S.
faecalis and S. faecium) has been suggested as
useful for indicating the presence of viruses,
particularly in sludge and seawater
• The fecal streptococci to fecal streptococci
ratio (FC:FS ratio) serves as an indicator of the
origin of pollution of surface waters
• A ratio of 4 or more indicates a contamination
of human origin, whereas a ratio below 0.7 is
indicative of animal pollution
• This ratio is only valid for recent (24 hours)
fecal pollution
Anaerobic Bacteria:
• Clostridium perfringenes is the main anaerobic
indicator bacteria
• Gram-positive, spore-forming, rod shaped
bacterium that produces spores
• Resistance to environmental stresses and to
disinfection
• The hardy spores make this bacterium too
resistant to be useful as indicator organisms
• As an indicator of past pollution and as a
tracer to follow the fate of pathogens
• To be reliable indicator for tracing fecal
pollution in the marine environment
Bifidobacteria:
• Anaerobic, non-spore forming, gram-positive
bacteria
• B. bifidum, B adolescentis, B infantis are
primarily associated with humans
Bacteriodes spp.:
• Anaerobic bacteria occurs in the intestinal tract
at concentrations in the order of 10 10 cells per
gram of feces
• Survival of B. fragilis in water is lower than
that of E. coli or S. fragilis
• A fluorescent antiserum was suggested as a
useful method for indicating the fecal
contamination of water
Bacteriophages:
• Are similar to the enteric viruses but are more
easily and rapidly detected in environmental
samples
• Found in higher numbers than enteric viruses
in wastewater and other environments
• Potential use of coliphages as water quality
indicators in estuaris, seawater, recreational
freshwater and potable water
• Coliphages exhibited the best correlation with
enteric viruses in polluted streams.
• The incidence of both enteric viruses and
coliphages were inversely correlated with
temperature
• Indicators for assessing the removal efficiency
of water and wastewater treatment plants.
• In an activated sludge system, coliphages that
formed plaques greater than 3mm were
significantly correlated with numbers of
enteroviruses
• The potential bacteriophage of Bacteriodes spp. to
serve as indicators of viral pollution
• Phages active against Bacteroides fragilis
HSP40were detected in feces (found in 10% of
human feces not in animal), sewage and other
polluted aquatic environment.
• Absent in non polluted water
• Do not multiply in environmental conditions
• More resistant to chlorine as compared to bacteria
(S. faecalis, E. coli) or viruses (poliovirus type 1,
rotavirus SA11 and coliphage f2)
• They are less resistant than coliphage f2 to UV
irradiation
• Thus these organisms may be suitable
indicators of human fecal pollution
• Their use makes possible the distinction
between human and animal fecal pollution
• Display a positive correlation with
enteroviruses and rota viruses
• Their persistence is similar to that of hepatitis
A virus in seawater
Yeast and Acid-fast Organisms:
• Proposed yeasts and acid- fast mycobacteri
(Mycobacterium fortuitum and M. phlei) as
indicator of disinfection efficiency
• Because the acid fast M. fortuitum is more
resistant to free chlorine and ozon than E. coli
or poliovirus type I
Heterotrophic Plate Count (HPC):
• HPC represents the aerobic and facultative
anaerobic bacteria that derive their carbon and
energy from organic compounds
• The number of recovered bacteria depend on
the media composition, period of incubation
(1-7 days) and temperature of incubation (20-
35oC)
• This group includes gram-negative bacteria
belonging to the following genera:
• Pseudomonas, Aeromonas, Klebsiella,
Flavobacterium, Enterobacter, Citrobacter,
Serratia, Acinetobacter, Proteus, Alcaligenes,
Moraxella
• The HPC microorganisms found in chlorinated
distribution water shown in the following
table:
• Some members of this group is opportunistics
pathogens (e.g., Aeromonas, Flavobacterium
etc)
• Less information about the effect of
heterotrophs on human health
• In drinking water the number of heterotrops
vary (104 CFU/ml or <500 CFU/ml potable
water)
• Number is mainly influenced by the
temperature, presence of residual chlorine, and
presence of assimilable organic carbon
• Found to be the most sensitive indicator for the
removal and inactivation of microbial pathogens in
wastewater
• Useful to water treatment plant operations with
regard to the following:
-- Assessing the efficiency of various treatment
processes including disinfection, in a water treatment
plant
-- Monitoring the bacteriological quality of the finished
water during storage and distribution
-- Determining bacterial growth on surfaces of
materials used in treatment and distribution systems
-- Determining the potential for regrowth or
aftergrowth in treated water in distributions
systems
Chemical Indicators of Water Quality
Free chlorine residue:
• Is a good indicator for drinking water quality
Levels of endotoxin:
• Endotoxins are lipopolysaccharides present in
the outer membrane of gram negative bacteria
• Their concentration in environmental samples
is conveniently measured by the Limulus
amoebocyte lysate (LAL) assay
• This test is based on the reaction of white
blood cells of the horseshoe crab with
endotoxin
• This reaction leads to an increase in sample
turbidity that is conveniently measured with a
spectophotometer
• There is a correlation of endotoxin levels in
wastewater and drinking water with the levels
of total and fecal coliforms
Detection of Indicator Microorganisms
Detection of Total and Fecal Coliforms:
• The total coliforms group includes all the
aerobic and facultative anaerobic, gram-
negative, non-spore-forming, rod-shaped
bacteria
• Ferment lactose with gas production within
48hrs at 35oC
• Total coliforms are detected by the most
probable numbers (MPN) technique or by the
membrane filtration methods
• These procedures are described in detail in
Standard Methods for the Examination of
Water and wastewater
• MPN generally overestimates coliforms
numbers in tested samples
• The overestimation depends on the number of
total coliforms present in the water sample and
the number of tubes per dilution
• Fecal coliforms are defined as those bacteria
that produce gas when grow in Lactose broth
at 44.5oC or produce blue colonies when grow
in m-FC agar at 44.5oC
• The traditional MPN five test is more useful.
• Several factors influence the recovery of
coliforms among them the type of growth
medium, the diluent and membrane used, the
presence of non coliforms and the sample
turbidity
Most probable number
• MPN use when turbidity exceeds 5
nephelometric turbidity unit (NTU)
• which measures the intensity of light scattered at
90 degrees as a beam of light passes through a
water sample.
• HPC bacteria able to reduce the number of
coliform bacteria presumably by competing
successfully for limiting carbon source
• Occurrence of injured bacteria
• Injury is due to physical (e.g., temperature,
light), chemical (toxic metals, organic toxicants
and chlorination) and biological factors
• Bactria do not grow well in the selective media
used (presence of selective ingredients such as
bile salts and deoxycholate) under temperatures
much higher than from environment
• In gram negative bacteria, injury cause damage
to the outer membrane which become more
permeable to selective ingredients such as
deoxycholate
• The injured cells can undergo repair when
grow on a nonselective nutrient medium
• The low recovery of injured coliforms in
environmental samples may underestimate the
presence of fecal pathogen in the samples
• Copper and chlorine induced have been
studied in vitro and in vivo conditions
• Injured pathogens display a temporary
reduction in virulence but under suitable in
vivo conditions they may regain their
pathogenicity
• Copper and chlorine stressed cells retain their
full pathogenic potential even after exposer to
the low pH of the stomach of orally infected
mice
• M-T7 agar used for the recovery of injured
cells
• Recovery generally improved when the
samples were preincubated at 37 degree C for
8hrs
• Chlorine stressed E. coli displays reduced
catalase leading to its inhibition from the
accumulated hydrogen peroxide
• Detection of chlorine stressed coliform
bacteria can also be accomplished by
incorporating catalase and/or pyruvate into the
growth medium to block and degrade
hydrogen per oxide
Membrane Filtration Method:
• Until, 1950s for indicator purposes, on the
enumeration of coliforms and E. coli based on the
production of gas from lactose broth in liquid
media
• And MPN using the statistical approach suggested
by McGrady
• In Russia and Germany, attempted to culture
bacteria on membrane filters
• In 1943, Mueller in Germany was using
membrane filters in conjunction with Endo broth
for the analysis of potable water
• Alternative to MPN
• Inability to gas production was considered a
major drawback
• For E. coli and the related coliforms were all
based upon cultural characteristics, including the
ability to produce gas from lactose fermentation
• The thermotolerant coliforms include Klebsiella,
E. coli as well as certain Enterobacter and
Citrobacter able to grow under the conditions
defined for thermotolerant coliforms
Advantages:
• Large volume of water
• Rapid
• Saving labour, amount of medium, less
glassware required
Disadvantages:
• Unsuitable for high turbid water
Standard Plate Count Methods:
• Usually 1.0 and 0.1 ml quantities of the water
sample are plated on an agar medium and
incubated for 24 h
• For plate count generally used selective and
differential media e.g. MacConkey, EMB etc
Rapid Methods for Coliform Detection:
Enzymatic Assays:
• Constitute an alternative approach for
detecting indicator bacteria
• Specific, sensitive and rapid
• The detection of total coliforms consists of
observing β-galactosidase activity, which is
based on the hydrolysis of the substrate o-
nitrophenyl-β-D-galactopyranoside (ONPG) to
yellow nitrophenol, which absorbs light at
420nm
• A fluorogenic compound, 4-
methylumbelliferone-β-D-galactoside can also
be used as a substrate for β-galactosidase
• Detection of total coliform can be improved by
incorporating isopropyl-β-D-
thiogalactopyranoside (IPTG), a gratuitous
inducer of β-galactosidase production in
growth medium
• Rapid assays for detection of E. coli are based
on the hydrolysis of a fluorogenic substrate 4-
methylumbelliferone glucuronide (MUG) by
β-glucuronidase, an enzyme found in E. coli
• The end product is fluorescent and can be
easily detected with a long-wave UV lamp
• Used for the detection of E. coli in clinical and
environmental samples
• Beta –glucuronidase is an intracellular enzyme
that is found in E. coli as well as some
Shigella spp
• Used in detection of E. coli in water and food
samples
• Colilert is commercial available
• A MUG-based solid medium was proposed for
the detection of E. coli after only a 7.5 hr
incubation
• Specificity 96.3%
• Chromogenic substrate, 5-bromo-4-chloro-3-
indoxy-β-glucuronide (BCIG) is potentially
useful for the rapid and specific identification
of E. coli
• E. coli colonies turns blue following
incubation of samples for 22-24 hrs at 44.5 oC
• 99% of the BCIG positive blue colonies were
also MUG positive
• Similar enzymatic methods have been
developed for the detection of fecal
streotococci
• Can be detected by incorporating a fluorogenic
substrate, 4-methylumbelliferone-β-
Dglucoside (MUD) into a selective medium
• Freshwater, wastewater and marine water
• The enterococci group can be rapidly detected
by a fluorogenic-chromogenic enzymatic
assay
• Based on the detection of the activity of two
specific enzymes, pyroglutamyl
aminopeptidase and β-D-glucosidase
• The enterococci group can be rapidly detected
by a fluorogenic-chromogeic enzymatic assay
• The test is based on the detection of the
activity of two specific enzyme, pyroglutamyl
aminopeptidase and β-D-glucosidase
Heterotrophic Plate Count (HPC):
• HPC in water and wastewater is defined as the
total number of bacteria that can grow following
incubation of the sample on plate count agar at
35oC for 48hrs
• These bacteria may interfere with the detection
of coliforms in water samples
• HPC is greatly influenced by the temperature
and length of incubation, the growth medium
and the plating method (pour plate versus
spread plate)
• A growth medium, designated R2A, developed
for use in heterotrophic plate counts
• The medium is recommended for use with an
incubation period of 5-7 days at 28 oC
• HPC should not exceed 500 colonies per 1ml
Detection of Fecal Streptococci:
• The following methods are commonly used for
the detection and estimation of the number of
fecal streptococci
a. i. Inoculation of multiple portion of water into
tubes of glucose azide broth
ii. Incubated at 37oC for 72 hrs
iii. Acidity is observed
iv. A heavy inoculum is further subcultured into
tubes of glucose azide broth
v. incubated at 45oC for 48 hours
vi. All tubes showing acidity at this temperature
contain fecal streptococci
b.
i. The inoculation of multiple portion of water
into tubes of buffered azide glucose glycerol
broth (BAGG) medium
ii. Incubated at 45oC for upto 48hrs
iii. Growth with the production of acid is almost
definite evidence of the presence of fecal
streptococci
iv. Confirmed by microscopic examination of
Gram staining
Membrane Filtration Technique:
• After filtration of water, membrane is placed
on a well dried plate of glucose azide agar or
KF- streptococci agar
• Incubated at 37oC for four hours or and then at
44oC or 45oC for 44 hours
• All red maroon colonies are counted as fecal
streptococci
Detection of anaerobic spore forming
organisms:
Detection and estimation of the number of spore of
C. perfringenes
i. At first the water samples are preheated at 75 oC
for 10 minutes to destroy all vegetative cells
ii. Inoculated the samples into differential
reinforced clostridial medium (DRCM) in
screw capped bottles
iii. The bottles should be filled up if necessary so
as to leave only a small air space
iv. A positive reaction will be shown by
blackening of the medium due to reduction of
the sulfite and precipitation of ferrous sulfide
v. A loopful from each positive bottle should be
subcultured into a tube of litmus milk that has
been freshly steamed and cooled
vi. Then incubated at 37oC for 48 hours
vii. C. perfringenes will produce a “stormy clot”
in which the milk is acidified and coagulated and
the clot disrupted by gas
A sulfite-reduction method using a solid
medium:
i. Volumes of water are mixed with the melted
medium tubes or in the petridishes
ii. When the medium has set, incubated at 37oC or
44oC for 24 to 48 hrs
iii. The black colonies in the depth of the medium
are counted
iv. The presence of black colonies of over 3mm in
diameter indicates contamination with C.
perfringenes
General Scheme of Laboratory Testing For Detection
of Coliform bacteria in water
Presumptive test
Inoculation into lactose
broth
No gas: coliforms are
Gas production:
not present,
presumtive
examination stop here
evidence of coliforms
Confirmed Tests:
Confirmed Test: transfer
made from gas producing
lactose broth
or
Brilliant green lactose- Eosin-Methylene Blue Agar
bile broth(BGLB): inhibit (EMB): E.coli produce small,
growth of lactose dark, black dark centers with green
fermenters other than metalic sheen colonies
coliforms. Gas formtation Enterobacter: large, pinkish,
is a confirmatory test mucoid colonies, dark centers
Completed test:
If gas present If typical colonies are present
Typical colonies are selected from EMB {if BGBL
used in confirmed test, the broth culture is first
streaked onto EMB to obtain colonies) and
inoculated into
Lactose broth Nutrient Agar media, cultural
Coliform produces gas characteristics, Gram staining
US EPA BDS
Parameters
standard for drinking water standard
TABC (CFU/100 ml) 1000 1000
TCC (CFU/100 ml) None None
E. coli (CFU/100 ml) None None
Color 15 (color unit) 5 (Haxen unit)
Odor 3 threshold odor number Unobjectionable
Taste Agreeable Agreeable
Turbidity (NTU) 0.5-1.0 5.0
pH 6.5-8.5 6.4-7.4
Salinity (ppt) 0.1 0.1
Turbidity (NTU) 0.5-1.00 5.0
TDS 500 500
Conductivity µs/cm NS NS
BOD (mg/l) 5.0 5.0
COD (mg/l) 40 40
Iron (mg/L) 0.3 0.30
Manganese (mg/L) 0.05 0.50
Zinc (mg/L) 5.00 3.00
Lead (mg/L) 0.015 0.01
Arsenic (mg/L) 0.01 0.05
Cadmium (mg/L) 0.005 0.003
• The drinking water of most communities and
municipalities comes from surface sources e.g.,
rivers, streams and lakes
• Natural waters are likely to be polluted with
domestic, agricultural and industrial wastes
• When the rain water passes through the
atmosphere gets dissolved oxygen, carbon
dioxide, other gases and floating dust
• The most common salts are the carbonates,
sulphates, chlorides and nitrate of calcium,
magnesium, sodium and potassium
• These impart alkalinity, hardness and salinity
• The impurites in water may be classified into
inorganic and organic
• May be present in the form of suspended,
colloidal and dissolved salts
• Living organisms in water includes
microorganisms, aquatic life and microscopic
form of life
• These impart turbidity, colour, bad taste, odour
etc
• Some bacteria causes disease
• The iron oxide and manganese impart red,
black or brown colour
• The limits for inland/raw surface water used
for municipal supply and bathing are
Parameters Limit
Coliform 5000 MPN /100 ml
pH 6 to 9
Flourides 1.5 mg/l
Chlorides 600 mg/ l
cyanide 0.01 mg / l
Selenium 0.05 mg / l
Lead 0.1mg/ L
Chromium 0.05 mg / l
Arsenic 0.2 mg / l
Dissolved Oxygen 40% saturation
5day 20oC BOD 3 mg/l
Phenolic compounds 0.001 mg/l
Testing of Water:
- Physical
- Chemical and
- Biological
Physical Testing:
Temperature:
• Temperature of water should not beyond 27 oC
for drinking purposes
Turbidity:
• Indication of apparent colour of water
• Due to the presence of suspended inorganic
matter like silt, clay etc
• Expressed by the amount of suspended matter
in ppm by weight in water as ascertained by
optical observation
• Turbidity measured by using standard silica
scale
• Also be measured by immersing a standard
platinum wire 1.0 mm in diameter in the sample
water under standard lighting conditions and
noting the exact depth at which it disappears
from view while seeing through naked eye
• Also measured by Jackson turbidity meter
• For less turbidity (5- 100ppm), the Bayles
turbidity meter is used
• Turbidity should be limited 10 units (ppm) on
the silica scale
Colour:
• Due to substances in the true solution or in
colloidal suspension
• Measuring by comparing water with standard
solutions of platinum-cobalt or standard coloured
glass discs
• The unit of colour is that produced by 1.0 mg of
platinum in a litre of distilled water
• Maximum permissible colour for drinking purposes
is 20 units or 20ppm or platinum cobalt scale
• Colour is harmless but more than 20ppm as the
consumers feel repellant
Taste and Odours:
• Due to mineral salts, tarry substances, industrial
wastes, municipal waste etc
• Expressed as a number which is called
“Threshold Odour Number”
• Number indicates the maximum dilution to be
made of a sample e.g., if 100 cm3 of fresh water
have to be added to 8cm3 of a sample of water
to produce no effect of odour in it, the
“Threshold Number is 8”
• The water should be served to the consumers
which is free of objectionable taste and odour
Chemical Examination:
Total Solids:
• Generally present in the range of 600-1500ppm
• TDS consists of chlorides, nitrates and
compounds causing hardness
• Permissible limit is 500-1000 ppm but
preferable less than 500ppm
• Suspended, dissolved and colloidal solids are
determined separately
• Suspended solids are obtained by filtering the
sample through a fine filter and weighing the
dried material on the filter
• The dissolved and colloidal solids are obtained
by evaporating the filtered sample of water
and weighing the residue
Chloride:
• Specially conducted for sodium chloride
• If the proportion of NaCl is more than the
specified limit, indicate the sewage
contamination
• Chloride may be determined by adding silver
nitrate solution of known concentration and
potassium chloride to a sample of water
containing
• The permissible limit of chloride 250ppm
• For the test, 100 ml of sample is taken for
titration
• It is placed on a porcelin basin
• One ml of potassium chromate solution
(prepared by dissolving 5g of potassium
chromate in distilled water and making upto
100 ml) is added and the sample is titrated
with standard silver nitrate solution
Hardness:
• The hardness of water is caused by
bicarbonates, carbonates, sulphates, chlorides
and nitrates of calcium and magnesium
• Is not unfit for drinking water unless the
hardness is excessive
• But it consume more soap in laundries and
forms deposits in the boilers
• Too soft water is tasteless
• The harness is usually expressed in ppm of
calcium carbonate presence in water
• It may be determines by soap test
• The standard soap solution is added in the
sample of water and the mixture is agitated
vigorously and five minutes are allowed for
leather formation the total hardness of water is
the sum of concentrations of all the metalic
cations except the alkalications
Dissolved Oxygen:
• The amount of oxygen present in water
absorbed by 10% solution of potassium
permanganate at 27 degree C for 4 hours
shows the quantity of a carbonaceous matter of
organic origin
• Should not be more than 10 ppm
Alkalinity:
• Caused by carbonates, bicarbonates and
hydroxide of sodium, calcium and magnesium
• Mostly the water are alkalines due to common
presence of salts
• Alkalinity makes water tasteful and helps in
coaggulation and does not affect pipes
• Excessive alkalinty interferes with coaggulation
• The alkalinity expressed in ppm in terms of
equivalent calcium carbonate
• Determined by titrating the sample against
standard acid using methyl orange indicator
Nitrogenous Compounds:
• These are like ammonia, nitrites and nitrates
• Presence indicates the organic contamination
• For test, known amount of water is boiled with
standard solution of potassium permanganate
which results in the breakdown of least stable
nitrogenous compounds and ammonia formed
• The amount of this ammonia which is called
albuminoid ammonia or albuminoid nitrogen
which should not be exceed 0.02ppm
• The nitrite and nitrates are formed due to
oxidation of organic matters
• Their presence indicates that the water was
polluted sometimes back
• Nitrite should be nil
• Nitrate should not be more than 20 ppm
• Ammonia should not be more than 0.15 ppm
Iron and Manganese:
• Usually present in water
• Are not objectionable if present upto 0.3 ppm
• Cause bad taste, colouration of clothes, floors,
utensils etc
• Also responsible for corrosion
• Manganese occurs in smaller amounts
• Also oxidized into a sedoments which
stimulates organic growth, clogs pipes and
cause discolouration of cloths
• The iron and manganese may be determined
by adding certain colouring reagents to the
sample of water and to the solution of known
amounts of iron and manganese
Soluble Mineral Impurities:
• All the natural water contain some percentage
of dissolved mineral matter
• All salts impart certain taste if present above
200ppm e.g., sodium chloride
• More than 300 ppm of NaCl is undrinkable
• Sulphate of sodium and magnesium if present
above 500ppm impart noticeable taste
• Recommended range is 500-1000 ppm
Residual Chlorine:
• Gives an indication that the bacteria are
reduced to a safe limit
• Determined by adding orthotolodin solution in
a sample of water
• The yellow colour of the sample ensures the
presence of residual chlorine
• Determined by adding potassium iodide and
starch solutions to a sample of water
• The blue colour appearing is neutralized by
titrating with 10% normal sodium thiosulfate
solution and the amount of chlorine is
examined
• The minimum amount of free chlorine should
be 0.05 to 0.10 ppm at all points in the
distribution system and 0.2 – 0.3 ppm during
water borne diseases
Sulphates:
• Should not be more than 250ppm
• Two methods for determining sulphates
a) Gravimetric method
- In which sulphates in the sample are
precipitated by barium chloride and the
precipitate is weighed as barium sulphates
b) EDTA method
- In which a measured excess of standard barium
chloride solution is added to the sample and the
excess barium chloride is estimated by the
titration against EDTA solution
