BACTERIOLOGY OF WATER AND ANALYSIS - BASICS BY Dr.T.V.Rao MD

1. BACTERIOLOGY OF WATER
AND ANALYSIS
basics
Dr.T.V.Rao MD
Dr.T.V.Rao MD @ Rao's Microbiology 1

2. The Quality of Water is polluted
with many Factors
Dr.T.V.Rao MD @ Rao's Microbiology 2

3. Bacteriology of water to identify the
bacterial contamination
•The bacteriological examination of water is
performed routinely by water utilities and many
governmental agencies to ensure a safe supply of
water for drinking, bathing, swimming and other
domestic and industrial uses. The examination is
intended to identify water sources which have
been contaminated with potential disease-causing
microorganisms.
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4. How the water gets contaminated
•Such contamination generally occurs either
directly by human or animal feces, or indirectly
through improperly treated sewage or
improperly functioning sewage treatment
systems. The organisms of prime concern are the
intestinal pathogens, particularly those that
cause typhoid fever and bacillary dysentery
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5. Common Diseases Spread by Water
•Typhoid fever
•Cholera
•Diarrhoeal diseases
•Polio myelitis
•Viral hepatitis A and E
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6. Contamination Happens
When water comes in Contact
•Faecal matter
• Sewage
contamination
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7. How the Water Contaminated
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8. Natural Water contaminated with
•Pseudomonas spp
•Flavobacterium
•Chromobacterium
•Acinetobacter spp
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9. Water we drink should be Free from
Should be free from pathogenic
bacteria
The primary criteria is the water
should be free from Coliforms if
they are present it indicates faecal
contamination
Thermotolerant E.coli bacteria
indicates the presence of
contamination
Faecal Streptococci thermos
tolerant indicates contamination
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10. Bacteriological Analysis of Water
• Bacteriological water
analysis is a method of
analysing water to
estimate the numbers of
bacteria present and, if
needed, to find out what
sort of bacteria they are.
It represents one aspect
of water quality.
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11. How the bacteriological analysis of water
Helps
• It is a microbiological
analytical procedure which
uses samples of water and
from these samples
determines the
concentration of bacteria. It
is then possible to draw
inferences about the
suitability of the water for
use from these
concentrations. Dr.T.V.Rao MD @ Rao's Microbiology 11

12. What are the Indicator organisms
contaminate water
• The common feature of all these routine screening
procedures is that the primary analysis is for indicator
organisms rather than the pathogens that might cause
concern. Indicator organisms are bacteria such as non-
specific coliforms, Escherichia coli and Pseudomonas
aeruginosa that are very commonly found in the
human or animal gut and which, if detected, may
suggest the presence of sewage
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13. Methods used in Culturing of Water
• Analysis is usually performed
using culture, biochemical and
sometimes optical methods.
When indicator organisms levels
exceed pre-set triggers, specific
analysis for pathogens may then
be undertaken and these can be
quickly detected (where
suspected) using specific culture
methods or molecular biology.
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14. Multiple tube method
• One of the oldest methods is called the multiple tube
method. In this method a measured sub-sample
(perhaps 10 ml) is diluted with 100 ml of sterile
growth medium and an aliquot of 10 ml is then
decanted into each of ten tubes. The remaining 10 ml
is then diluted again and the process repeated. At the
end of 5 dilutions this produces 50 tubes covering the
dilution range of 1:10 through to 1:10000.
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15. Methodology of Bacterial analysis of
Water
• The tubes are then incubated at a pre-set temperature for a specified
time and at the end of the process the number of tubes with growth
in is counted for each dilution. Statistical tables are then used to
derive the concentration of organisms in the original sample. This
method can be enhanced by using indicator medium which changes
colour when acid forming species are present and by including a tiny
inverted tube called a Durham tube in each sample tube. The Durham
inverted tube catches any gas produced. The production of gas at 37
degrees Celsius is a strong indication of the presence of Escherichia
coli.
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16. Laboratory pictures show how the Water is
Analysed
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17. Membrane filtration
• Most modern laboratories use a refinement of total
plate count in which serial dilutions of the sample are
vacuum filtered through purpose made membrane
filters and these filters are themselves laid on nutrient
medium within sealed plates. The methodology is
otherwise similar to conventional total plate counts.
Membranes have a printed millimetre grid printed on
and can be reliably used to count the number of
colonies under a binocular microscope.
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18. Pour plate method
•When the analysis is looking for bacterial species
that grow poorly in air, the initial analysis is done
by mixing serial dilutions of the sample in liquid
nutrient agar which is then poured into bottles
which are then sealed and laid on their sides to
produce a sloping agar surface. Colonies that
develop in the body of the medium can be
counted by eye after incubation.
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19. Pour plate method
•The total number of colonies is referred to
as the Total Viable Count (TVC). The unit of
measurement is cfu/ml (or colony forming
units per millilitre) and relates to the
original sample. Calculation of this is a
multiple of the counted number of colonies
multiplied by the dilution used.
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20. PLATE COUNT
• Test of water contamination in which the number of the
colonies of coliform-bacteria Escherichia coli (E. coli) per 100
milliliter of water is counted. The result is expressed as
'Coliform Microbial Density' and indicates the extent of fecal
matter present in it.
• According to common water quality standards drinking water
must be completely free from any colony, bathing and
swimming pool water can have about 200 colonies, and
recreational (fishing and boating) water about 1000 colonies.
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21. Multiple tube Methods
• Prior to the membrane filter
method, the multiple tube
method was used for
bacteriological examination of
water. In conjunction with this
test, a total plate count was also
conducted. These tests are
discussed here since they are
still considered standard tests in
the water works field.
Dr.T.V.Rao MD @ Rao's Microbiology 21

22. Dr.T.V.Rao MD @ Rao's Microbiology 22

23. Reporting the Results of Water
Analysis
•When a water sample
arrives at the
laboratory, two tests,
the plate count and
the coliform test by
the multiple tube
method, are made and
reported to the
operator. Dr.T.V.Rao MD @ Rao's Microbiology 23

24. Reporting the Results of Water Analysis
• The coliform test actually
consists of two steps known as
the presumptive test and the
confirmed test. Under certain
conditions, it is necessary to go
one step further and make a
completed test; however, this
step is not always necessary. To
make the tests, small portions of
the water sample are used in
accordance with the following
procedures.
Dr.T.V.Rao MD @ Rao's Microbiology 24

25. The Plate Count
• The plate count is a test made by
the laboratory to determine the
total number of bacteria present in
the sample. This test does not
differentiate between the many
different types of bacteria and is
thought of as giving index to the
general "housekeeping" practices.
A "high" count indicates that some
type of contamination is present
and is undesirable.
• The test is made by placing a
portion of agar in a petri dish.
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26. Making the Microbes to grow on the
Petri dish
• A portion of the water sample is
placed in the petri dish along
with the agar. It is then placed
in an incubator with the
temperature at 37°C or 98.6°F,
which is body temperature.
After 24 hours, the plate is
removed, examined, and the
colonies in and on the agar are
counted and recorded on the
report form as "Bacteria per ml
at 37°C".
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27. Types of Nutrient media used in
analysis
•MacConkey agar is culture medium designed to
grow Gram-negative bacteria and stain them
for lactose fermentation. It contains bile salts
(to inhibit most Gram-positive bacteria), crystal
violet dye (which also inhibits certain Gram-
positive bacteria), neutral red dye (which stains
microbes fermenting lactose), lactose and
peptone.
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28. Presence of Bacteria Means
• The presence of the Coli-
aerogenes group of bacteria in
the above tests does not
definitely mean that harmful
bacteria are present. Coliform
bacteria are normally present in
great numbers in the human
intestine and, except in unusual
circumstances, are not harmful
to humans.
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29. What the Indication of presence of
Bacteria
• When present in a water
sample, they do, however,
indicate the presence of faecal
contamination and the
possibility that harmful
(pathogenic) organisms, such as
typhoid fever germs, may be
present. Therefore, the tests are
not measures of actual disease-
producing organisms, but rather
are indicators of the possibility
that they are present.
Dr.T.V.Rao MD @ Rao's Microbiology 29

30. Public Health Concept
•The coliform group has
been used extensively
as an indicator of
water quality and has
historically led to the
public health
protection concept.
Dr.T.V.Rao MD @ Rao's Microbiology 30

31. Popular Methods to analyse the
Water
• Routine and widely accepted techniques are discussed, as
are methods which have emerged from recent research
developments. Approved traditional methods for coliform
detection include the multiple-tube fermentation (MTF)
technique and the membrane filter (MF) technique using
different specific media and incubation conditions. These
methods have limitations, however, such as duration of
incubation, antagonistic organism interference, lack of
specificity and poor detection of slow-growing or viable but
non-culturable (VBNC) microorganisms.
Dr.T.V.Rao MD @ Rao's Microbiology 31

32. Methods in use for Bacteriological analysis
• The simple and inexpensive membrane filter technique is the most
widely used method for routine enumeration of coliforms in drinking
water.The detection of coliforms based on specific enzymatic activity
has improved the sensitivity of these methods. The enzymes beta-D
galactosidase and beta-D glucuronidase are widely used for the
detection and enumeration of total coliforms and Escherichia coli,
respectively. Many chromogenic and fluorogenic substrates exist for
the specific detection of these enzymatic activities, and various
commercial tests based on these substrates are available
Dr.T.V.Rao MD @ Rao's Microbiology 32

33. Bacteriological Identification with
molecular methods
• The immunological, polymerase chain reaction (PCR) and in-
situ hybridization (ISH) techniques. In the immunological
approach, various antibodies against coliform bacteria have
been produced, but the application of this technique often
showed low antibody specificity. PCR can be used to detect
coliform bacteria by means of signal amplification: DNA
sequence coding for the lacZ gene (beta-galactosidase gene)
and the uidA gene (beta-D glucuronidase gene) has been
used to detect total coliforms and E. coli, respectively.
Dr.T.V.Rao MD @ Rao's Microbiology 33

34. Is the PCR is precise in analysis of
Water
• However, quantification with PCR is still lacking in
precision and necessitates extensive laboratory work.
The FISH technique involves the use of
oligonucleotide probes to detect complementary
sequences inside specific cells. Oligonucleotide probes
designed specifically for regions of the 16S RNA
molecules of Enterobacteriaceae can be used for
microbiological quality control of drinking water
samples
Dr.T.V.Rao MD @ Rao's Microbiology 34

35. Detection of Virus
• Methods are available for isolation of Enterovirus and
other cytopathogenic viruses from water
• But are not part of routine tests unless epidemics
happens
• However Viruses are destroyed with Chlorination of
water
• The Free residual chlorination is at least 0.5 mg per
litre for a contact period of 30 minutes at ph. below 8
Dr.T.V.Rao MD @ Rao's Microbiology 35

36. Protozoa in Water
• Endamoeba histolytica
• Giardia species
• Balantidium coli
• However there are no specific
tests
• Coliforms are not reliable as
indicators of protozoal
contamination
Dr.T.V.Rao MD @ Rao's Microbiology 36

37. This Program is made for basic understanding on
Water bacteriology, for Undergraduate Medical
students Specific Protocols to be followed form your
Laboratory
Email
doctortvrao@gmail.com
Dr.T.V.Rao MD @ Rao's Microbiology 37