This document discusses methods for analyzing water samples, including bacteriological and chemical analysis. It describes three main methods for bacteriological analysis: presence-absence testing, most probable number testing, and membrane filtration. It also discusses various chemical testing methods like test strips, colorimeters, and specific kits for chemicals like arsenic. Solid phase extraction is introduced as a sample preparation technique that can be used prior to chemical analysis.

1. Submitted by:
Sharath. H.N
M Pharma 1st year
Bacteriological and chemical
analysis of Water

2. Contents
• Introduction to Analysis of water
• Bacteriological Analysis of Water
• Chemical analysis of water
• Solid phase extraction

3. • One of the things which make Earth a unique planet
in this universe is continuous availability of water, a
vital
• requisite for the existence of life. Water is also the
essential prerequisite of agriculture and industrial
production, the source
• of food needed for the survival of life. Thus, life on
earth is entirely and exclusively dependent on water.

4. • Water-related diseases continue as a major health
problem globally. An estimated, four billion cases of
diarrhea yearly represented 5.7% of the worldwide
disease in the year 2000 (WHO, 2002).
• So Physical , chemical and bacteriological analysis of
water was of great need for beneficity of people.

5. WHO Definition
Defines safe drinking water as water that “does not
represent any significant risk to health over the
lifetime of consumption, including different
sensitivities that may occur between life stages.”

6. 6
Categories of Contaminants
Chemicals
Microbiological
Physical

7. 7
Categories of Contaminants
Chemicals
 Organic
 Inorganic
 pH
Microbiological
Bacteria
Virus
Protozoa
Helminths
Physical
 Turbidity
 Colour
 Odor
 Taste

8. Water Sampling
-Microbiological sampling
-Indicator organisms for pathogen presence
Physical sampling
-turbidity, conductivity, total dissolved solids etc
Chemical sampling
- pH, dissolved oxygen, phosphates, chemical oxygen
demand, biological oxygen demand, mineral impurities
(iron, manganese, chloride, lead, sodium etc)

9. MICROBIOLOGY OF
WATER

10. Pathogens
• 4 types of pathogens
– Bacteria
– Virus
– Protozoa
– Helminths
bacteriaa on the tip of a pin
micro-organism that cause disease

11. FACTORS AFFECTING NUMBER AND TYPE OF
BACTERIA IN WATER
•Type of water:
Surface or deep
Mineral springs
•Presence of organic matter
•Temperature
•Light
•pH
•Dissolved oxygen
•Rainfall
•Season
•Storage
•Filtration

12. Microbiological Testing Methods
• 3 methods to determine presence of bacteria
in water:
– Presence-Absence (P-A)
– Most Probable Number (MPN)
– Membrane Filtration

13. Presence-Absence (P-A)
• Simplest method
• Add water sample to a bottle containing broth and let
it sit for 24-48 hours
• Color will change if indicator organism is present
• Does not show numbers of bacteria!
• If the sample is positive, the water should be re-tested
using membrane filtration to determine the number of
bacteria
• Not recommended by WHO for analysis of surface
water and untreated community water supplies
• Not recommended for testing the efficiency of
household water treatment technologies (e.g. biosand
filter)

14. Presence-Absence
Positive for
E. coli
Negative
Positive for
Coliforms

15. Most Probable Number (MPN)
• Tells the number of bacteria that are most likely to be
in the water sample
• Add water sample or diluted sample to 5 or 10 or test
tubes (or larger tray 50 – 96 tubes)
• Incubate for 24-48 hours
• Gas production and/or cloudiness will be visible if the
indicator organism is present
• Using a table provided, report the number of positive
tubes as number of colonies per 100 mL of sample.
• Typically used for wastewater or turbid samples

16. Most Probable Number (MPN)
# Positive
Tubes
MPN Index
(CFU/100mL)
0 <1.1
1 1.1
2 2.2
3 3.6
4 5.1
5 6.9
6 9.2
7 12.0
8 16.1
9 23.0
10 >23.0
Sample Table for
10 tube test

17. Most Probable Number (MPN)
• Characteristics:
– Quantitative results
– Simple to understand and use
– Relatively inexpensive
– Can be used with turbid water
– More labor-intensive than P-A
– Requires some training
– Requires incubator & other equipment

18. Membrane Filtration
• Most accurate method to count bacteria
• Filter 100 mL of a water sample
– Add broth to a Petri dish which provides nutrients for
the indicator organism to grow
– Filter the water using the filtration equipment
– Transfer the filter paper to the Petri dish
– Incubate for 24-48 hours depending on the broth
• If the indicator organism is present, colonies will appear
on the filter paper and can be counted
• Results are reported as the number of colonies per 100
mL of water sample (CFU/100mL)
– CFU = colony forming units

19. 19
Membrane Filter Technology
• A membrane is a thin material that has pores (holes)
of a specific size
• Membranes trap larger particles that won’t fit through
the pores of the membrane, letting water and other
smaller substances through to the other side

22. Membrane Filtration Equipment
Field Kits

23. 23
Membrane Filtration Equipment
Incubator

24. 24
Membrane Filtration Equipment
Nalgene Testing Kit

25. 25
Membrane Filtration
• Advantages:
– Able to count the number of bacteria
– Most accurate test method
– Ability to test many samples at once
– Internationally recognized method
– Rapid
– Easy & Economical.
– Gives direct result.
– Useful in rural areas.
– Samples can be tested in the field

26. 26
Membrane Filtration
• Limitations:
– More labour intensive than MPN, P-A
– Requires more training
– Requires additional equipment
– Cost of consumables can be high in many countries
– Turbid water interferes with bacterial growth.
– Noncoliforms interferes with counting of coliforms.
– Toxic substances in the water may be absorbed by filter and interferes
with bacterial growth.

27. 27
Chemical testing of water
• Chemical Testing Methods
– Test strips
– Colour disc comparators
– Colorimeters & photometers
– Digital meters
– Arsenic specific kits

28. 28
Chemical Tests
• There are many different chemicals that can be
found in our drinking water
• Difficult and expensive to test for all chemicals so we
need to select a few that are a priority in the local
area
– Iron, Manganese
– Arsenic, Fluoride
– Chlorine
– Total Dissolved Solids

29. 29
Chemical Test Methods
• Test (reagent) strips
• Colour disc comparators
• Colorimeter and photometer
• Digital meters

30. 2009-08 30
Test (Reagent) Strips
• Designed to react with
specific chemicals
– pH, chlorine, hardness, etc.
• Compare colour on
stick to colour chart
Advantages:
– Inexpensive
– Easy and simple
– Provides rough estimate
Limitations:
– Requires visual interpretation of
colour
– Low accuracy +/- 10 %

31. 31
Colorimeters & Photometers
Photometer
(Wagtech)
• Uses light source to
measure chemical
concentration
• Test a range of chemicals
Advantages:
– More accurate
Limitations:
– More expensive
– Power source necessary
– Proper training required
Colorimeter (HACH)

32. 32
Arsenic Test Kits
• Designed specifically for
arsenic
Advantages:
– Fairly accurate – range 2 to
100 ug/L
– Portable
– Relatively easy to use
Limitations:
– Requires visual interpretation
of colour
– Expensive

33. Solid phase extraction
Solid phase extraction (SPE) is an increasingly useful sample
preparation technique. With SPE, many of the problems
associated with liquid/liquid extraction can be prevented, such as
incomplete phase separations, less-than-quantitative recoveries,
use of expensive, breakable specialty glassware, and disposal of
large quantities of organic solvents.
SPE is more efficient than liquid/liquid extraction, yields
quantitative extractions that are easy to perform, is rapid, and can
be automated. Solvent use and lab time are reduced.

34. SPE is used most often to prepare liquid samples and
extract semivolatile or nonvolatile analytes, but also can be
used with solids that are pre-extracted into solvents.
SPE products are excellent for sample extraction,
concentration, and cleanup. They are available in a wide
variety of chemistries, adsorbents, and sizes.
Selecting the most suitable product for each application
and sample is important.

35. Based on types of seperations solid phase extraction
was classified into 4 types
•Reversed Phase
•Normal Phase
•Ion Exchange
•Anion exchange
•Cation exchange

36. Reversed Phase SPE
Reversed phase separations involve a polar or moderately polar sample
matrix and a nonpolar stationary phase.
The analyte of interest is typically mid- to nonpolar. Several SPE materials,
such as the alkyl- or aryl-bonded silicas (LC-18, ENVI-18, LC-8, ENVI-8,
LC-4, and LC-Ph) are in the reversed phase category.
Here, the hydrophilic silanol groups at the surface of the raw silica packing
(typically 60Å pore size, 40μm particle size) have been chemically modified
with hydrophobic alkyl or aryl functional groups by reaction with the
corresponding silanes.

37. Normal Phase SPE
Normal phase SPE procedures typically involve a polar analyte,
a mid- to nonpolar matrix (e.g. acetone, chlorinated solvents, and
hexane), and a polar stationary phase Polar-functionalized bonded
silicas (e.g. LC-CN, LC-NH2, and LC-Diol), and polar adsorption
media (LC-Si, LC-Florisil, ENVI-Florisil, and LC-Alumina) typically
are used under normal phase conditions.
Retention of an analyte under normal phase conditions is primarily due to
interactions between polar functional groups of the analyte and polar
groups on the sorbent surface.

38. Ion Exchange SPE
Ion exchange SPE can be used for compounds that are charged when in a
solution (usually aqueous, but sometimes organic).
Anionic (negatively charged) compounds can be isolated on LC-SAX or
LC-NH2 bonded silica cartridges. Cationic (positively charged) compounds
are isolated by using LC-SCX or LC-WCX bonded silica cartridges.
The primary retention mechanism of the compound is based mainly on the
electrostatic attraction of the charged functional group on the compound to
the charged group that is bonded to the silica surface.

39. Anion Exchange SPE
The LC-SAX material is comprised of an aliphatic quaternary
amine group that is bonded to the silica surface.
A quaternary amine is a strong base and exists as a positively-
charged cation that exchanges or attracts anionic species in the
contacting solution — thus the term strong anion exchanger
(SAX

40. Cation Exchange SPE
The LC-SCX material contains silica with aliphatic
sulfonic acid groups that are bonded to the surface.
The sulfonic acid group is strongly acidic (pKa <1), and
attracts or exchanges cationic species in a contacting
solution – thus the term strong cation exchanger (SCX).

41. References:
•International Journal of Innovative research in
Science, Engineering and Technology
•WHO Guidelines
•SUPELCO Pharma bulletein
•Internet source