2. Water Significance and Contamination
Water is essential for all forms of
life and certain human activities.
In the course of survival, certain
living organisms may contaminate
water required for use by others.
Human activities may also lead to
contamination of water sources
with toxic substances.

3. Why control water standards?
Health and survival of man and other
organisms depends on the purity of the
water they use.
Different measures are used to access and
control water quality with varying degrees
of relevance and acceptability.
Hence the need to adopt generalized
methods of assessment and arbitrary
standards for water quality.

4. Water Quality Assessment
Water
Quality
Physical Chemical Microbial

5. Water Quality Parameters
• Turbidity
• Palatability
• Conductivity
• Total Dissolved Solutes
• Organoleptic Properties
• etc
Physical
• pH
• Dissolved Oxygen Concentration
• Residual Free Chlorine
• Radionuclides
• Organic/ inorganic Chemical Contents
• etc
Chemical
• Faecal Coliform Count
• Total Coliform Count
• Faecal Streptococci
• Cysts/ Ova of Parasites
Microbial

6. Microbiological Analysis
Offers the most sensitive test for the
detection of recent and potentially
dangerous faecal pollution
Provides a hygienic assessment of water
quality with high sensitivity and
specificity
It is important to examine water
sources frequently by simple tests
rather than infrequently by more
complicated test or series of tests

7. Indicator Organisms
Investigation for individual specific
pathogens is rarely practical since they
are fewer in numbers than the non-pathogenic
organisms and their
detection methods are too demanding.
 Therefore, indicators of human/animal
pollution e.g. coliforms are used to
access water quality.

8. Indicators
E. coli
Faecal Coliforms
Total Coliforms
Faecal Streptococci

9. Indicator Organisms Cont’d
Escherichia coli
•Found in human and
animal faeces; in
sewage, treated
effluents, and natural
waters subjected to
faecal contamination
•Up to 109 per gram in
fresh faeces.
•Routine identification
is complex, but tests are
available for rapid
reliable identification.
Faecal coliforms
•Genus Escherichia and
some spp of Klebsiella,
Enterobacter and
Citrobacter
•May originate from
organically enriched
water or from decaying
matter.
•Rarely regrow in the
distribution system

10. Indicator Organisms Cont’d
Total Coliforms
•Gram-ve, bacilli tolerant to
bile salts.
•Not necessarily faecal
bacteria.
•Should not be detected in
treated water and, if found,
suggest inadequate
treatment, post treatment
contamination, or excessive
nutrients.
• Test used as an indicator
both of treatment efficiency
and of the integrity of the
distribution system.
Faecal streptococci
• Present in the faeces human
and animal faeces.
•Belong to the genera
Enterococcus and
Streptococcus
•Most spp are of faecal origin
and indicate faecal
contamination.
•Rarely multiply in polluted
water, and are more
persistent than the coliforms.
•Indicators of treatment
efficiency.

11. Sampling Sampling from a tap or pump
outlet
 Remove any attachments from tap that
may cause splashing.
 Wipe off the dirt from outside the tap.
 Turn on the tap at maximum flow rate
and let the water flow for 1-2 minutes.
 Sterilize it for a minute with flame
using gas burner, lighter or ignited
cotton wool soaked in spirit.
 Open the tap and allow water to flow
at medium rate for 1-2 minutes.
 Open the container for collecting the
sample and fill the water by holding
the bottle under the water jet. Leave a
small airspace to facilitate shaking at
thetime of inoculation prior to
analysis.
 Stopper the cap and label the
container.
Sampling from reservoir
 Submerge the bottle in the
water.
 Open the bottle inside of the
water.
 Fill it by holding it by the lower
part, submerging it to a depth
of about 30 centimetres, with
the mouth facing slightly
upwards. If there is a current,
the bottle should face the
current.
 Pull it out when the bottle is
filled.
 Discard a little water to provide
airspace
 Stopper the bottle and label it.

12. Methods for Microbial Analysis
Microbial
Analysis
Membrane
filtration
method
Multiple-tube
method

13. Sampling cont’d
Sampling from a dug well
 Attach a stone of suitable size to the sampling bottle with a
piece of string.
 Tie a 20 meter length of clean string on the bottle and to a
stick.
 Open the bottle as described above and lower into the well.
 Immerse the bottle completely in water without touching
the sides of the well and lower it down to the bottom of the
well.
 Pull it out when the bottle is filled.
 6. Discard a little water to provide airspace.
 7. Stopper and label the bottle.

14. Membrane Filtration Method
Aseptically introduce sample
into sterile membrane filter
assembly ( 0.2 or 0.45 μm)
Filter retains indicator
organisms
Transfer to a suitable culture
medium and incubate
Count colonies and report
results in CFU /100 ml

15. Membrane filtration machine

16. Multiple-tube method
Add different measured sample
volumes of to tubes containing a
suitable culture medium
Organisms reproduce and
produce acid with or
without gas
Statistically determine MPN of
organisms in the original
sample

17. Principle
 Separate analyses are usually conducted on five portions of each
of three serial dilutions of a water sample.
 The individual portions are used to inoculate tubes of culture
medium that are then incubated at a standard temperature for a
standard period of time.
 The presence of coliforms is indicated by turbidity in the culture
medium, by a pH change and/or by the presence of gas.
 The MPN index is determined by comparing the pattern of
positive results (the number of tubes showing growth at each
dilution) with statistical tables.
 The tabulated value is reported as MPN per 100 ml of sample.

18. Culture media
Medium uses Incubation periods
Lactose broth Total or thermotolerant
Isolation media
Double strength broth: Dissolve 71.2 g of lauryl sulphate broth in 1 litre distilled
water.
 After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
 Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
Single strength broth: Dissolve 35.6 g of lauryl sulphate broth in 1 litre distilled
water.
 After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
 Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
coliforms
48 hours at 35 ± 0.5 °C or 37
± 0.5 °C for total coliforms
and 24 hours at 44 ± 0.25 °C
or 44.5 ± 0.25 °C for
thermotolerant coliforms
MacConkey
Broth & Lauryl tryptose
(lactose) broth
Total or
thermotolerant
coliforms
48 hours at 35 ± 0.5 °C or 37
± 0.5 °C for total coliforms
and 24 hours at 44 ± 0.25 °C
or 44.5 ± 0.25 °C for
thermotolerant coliforms

19.  Prepare the required number of tubes of culture medium.
The volume and strength (single or double) of medium in
the tubes will vary depending on the expected
bacteriological density in the water and the dilution series
planned.
 Select and prepare a range of sample dilutions
 Pipette the appropriate volumes of sample and diluted
sample into the tubes of medium
 Label the tubes with the sample reference number, the
dilution and the volume of sample (or dilution) added to
the tube.
 Shake gently to mix the sample with the medium. Place the
rack in an incubator or water-bath for 48 hours at 35 ± 0.5
ºC or 37 ± 0.5 ºC.

20. Procedure cont’d
 After 18 or 24 hours, note which tubes show growth.
 Tubes that show turbidity and gas production, or a colour change
indicating the production of acid (if the medium contains a pH
indicator), are regarded as positive
 Return the tubes to the incubator and re-examine after a total of 48
hours of incubation. Continue with the next step of the procedure.
Confirmatory test
 Prepare the required number of tubes of confirmation culture medium
(BGLB broth for total coliforms and E. coli medium for faecal
coliforms).
 Using a sterile wire loop, transfer inocula from positive tubes into the
confirmation medium
 Incubate them for 48 hours at 35 ± 0.5 °C or 37 ± 0.5 °C for total
coliforms (BGLB broth) or for 24 hours at 44 ± 0.5 °C for faecal
coliforms (E. coli medium)

21. Procedure cont’d
 After the prescribed incubation time, note which tubes
show growth with the production of gas,
and record the number of positives for each sample
dilution
 The test organism is cultured in a
medium which contains
tryptophane, kovac’s reagent
added to confirm indole
production
 Compare the pattern of positive results with a most
probable number table such as one of those given in Tables

22. Typical sample volumes and number of tubes for multiple fermentation tube
analysis
Sample 50ml 10ml 1ml 0.1 0.01ml
Treated
drinking-water
1
1 5
Partially
treated
drinking
water
5 5 5
Recreational
water
5 5 5
Protected
source water
5 5 5

23. Membrane Filtration...
Method involves
comparatively
simpler
procedures.
Technique is
inappropriate for
highly turbid
waters.
Filters may be
expensive in
some countries.

24. MPN statistical table
Combination of
positive
MPN index /100ml 95% confidence level
Upper lower
0 0 0 < 2 - -
0 0 1 2 1 10
1 1 0 4 1 15
2 1 0 7 2 20

25. Multiple-tube method...
Method is applicable
to all kinds of water:
clear, coloured, or turbid
containing sewage, sludge,
mud or soil particles,
provided the contaminants
in the prepared test samples
are homogeneously
Distributed.
Method is
comparatively
complex.

26. Water Quality Standards
Regulations on the purity of water
used for various purposes.
Mandatory limits concerning
constituents and contaminants of
water known to be hazardous
and/or cause inconveniencies.
Sets of procedures and practices
required to meet the mandatory
limits.

27. Water Quality Standards
Water Quality
Standards
Surface Water
Quality
Standards
Drinking
Water Quality
Standards

28. Water Quality Parameters
Physical properties
Chemical / Inorganic Substances
Organic Constituents
Disinfectants and their by-products
Radionuclides
Microbiological Parameters.

29. ISO Drinking Water Quality Standard

30. Quality Standards of Water( Summary)
Physical
parameter
Fresh water Communal
water
Trout water High quality
water
Turbidity 50/25 NTU 10 NTU < 10 NTU
Temperature RT RT RT RT
Hardness 100mg/l cacO3
Chemical
parameter
Fresh water Communal
water
Trout water High quality
water
pH 6-9 6.8-8.5 7
Salinity N N N N
Chlorine 230mg/l 250mg/l N
Alluminum 87ug/l 6500ug/l
Dessolved
Not less than
Not less than
oxygen
5.mg/l
6mg/l
Not less than
6mg/l

31. Quality Standards of Water cont’d
Bacteriological
Fresh water Communal water High quality
parameter
water
Enterococcus 35org. /100ml
Feacal coliforms 200org. /100ml
Key:
NTU- Neplelometric Tubidity Unit
RT- Room Temperature
N- Normal
Refer to Table 1. Microbial Examination of Water and Water Quality Standards (group
2) for more detail