Pure Appl. Bio., 2(3): 96-103, Sep- 2013.
Research Article
Biochemical and microbiological evaluation of the water samples collected from different areas of district
Kohat and Mohamand Agency, Pakistan.
Ayaz Ali1, Jauhar Shah1, Farman Ullah1, Waheed Ullah1, Zia Urehman2, Nawab Ali1*
1
Department of Biotechnology & Genetic Engineering, Kohat University of Science and Technology, Kohat-26000, Khyber Pakhtunkhwa,
Pakistan.
2
PCSIR laboratories complex, Peshawar, Pakistan.
Corresponding author: Email: * nawabali_1857@yahoo.com
ABSTRACT
Contamination of drinking water sources mainly due to microorganisms is the major problem in many areas of
Pakistan. Pakistan is also facing the problem of contamination of drinking water which greatly affects human
health and quality of life. The most important component of human beings for living is water. Therefore, it is
important to analyze drinking water quality mostly in developing countries as the local people are mostly
unaware of the water pollution. In this study, twenty three samples of water were analyzed during a 3-month
period from the well and lake water supplies of different areas of Kohat and Mohamand Agency. The
bacteriological evaluation was done and several tests were performed such as Total Plate Count, Coliform,
Feacal coliform, Escherichia coli (E. coli) and Biochemical test. In this study, thirteen samples were in the
normal range and 10 samples were out of safety ranges fixed by World Health Organization (WHO). The
water which was not fit for drinking can be a consistent risk of the infectious diseases and continuous
assessment and purification strategies should be developed in these areas to reduce the microbial
contamination. The proper training by the local public authorities is required to educate the local community
about water pollution, their causes and preventive measures in order to improve the health status of the people
in the regions.
Key words E. coli and Biochemical test, Faecal coliform, Total coliform
INTRODUCTION
of industrial wastes and is a major problem in
Water is the most important component for living
Pakistan [4]. The main sources of drinking water in
things and to support the life processes. Around
Pakistan are tube wells and dug wells. Water gets
75% of the human body weight is composed of
polluted when its condition or composition or both
water. Without this it is not possible to maintain
are changed. The major sources of pollution in
life on this planet. The quantity of water present on
natural water are discharge of domestic and
earth is approximately 1.4 trillion cubic meters [1-
industrial wastes from urban, rural and industrial
3]. Due to increase in environmental pollution,
areas. Kohat and Mohamand agency are important
water resources are contaminated, which is a big
areas of Khyber Pukhtoonkhwa (KPK); therefore
hazard to life. This hazard is by regular discharge
process of development is also fast. The population
96
Ayaz et al
of these areas is constantly increasing. The increase
transmitted through polluted water. Cancer is also
in population give rise to many serious problems,
caused by the assembly of certain materials carried
especially increase in environmental pollution. As
by water to human organs [13-16]. Escherichia coli
a result the sources of water are being polluted
is a unicellular microorganism, associated with
constantly [5]. Drinking water must be free from
faecal contamination of water. In human faeces,
turbidity, color and odor as well as from
95% of coliforms are Escherichia coli [17-20]. For
objectionable
determination of water contamination and its
taste
which
have
different
inauspicious physiological effects [6-8].
standards the total aerobic plate count (TAC) is
Fecal coliform bacteria include Total coliform and
used as an indicator [21]. TAC is a subgroup of
microbiological
E.coli used for the indication of faecal pollution of
parameters of water quality. Water contamination
contaminated water [22]. The use of bottled water
indicates the presence of E. coli either by fecal
is being increasing from the past 30 years
material of humans or other warm-blooded
worldwide [23]. The consumer use bottle water
animals. According to U. S. Environmental
mainly due to safety and potential health benefits
Protection Agency (EPA), E. coli is a good
because bottled water is much safer than tape water
indicator of fecal pollution than fecal coliform to
[24, 25]. In Pakistan, rural populations are still
evaluate fresh water quality [9-11]. Contaminated
deprived of clean drinking water and in urban areas
water has direct and indirect effect; direct effect is
contamination of drinking water is due to leakage
on human health while indirect effect is through
in distribution systems [26-30].
consumption of foodstuffs being irrigated with
The aim of the study was to investigate the
sewage and industrial effluents. World Health
microbial contamination in the water samples
Organization estimated that around 80% of the
collected from different areas of Kohat and
population is deprived of pure drinking water in
Mohmand agency. This study will help to specify
developing countries and every year around 5
the areas where water is contaminated and
million people die as a result of illness causes due
purification
to unsafe drinking water. The major pollutants
accordingly by the local authorities.
present in water are mainly heavy metals,
Methods and Materials
microorganisms, fertilizers and other toxic organic
Water Sample Collection
compounds [12].
Sample collection was performed from February to
E.
coli
According
are
to
the
important
World
Health
strategies
should
be
applied
Organization
April (2013) and sampling was done randomly
estimation every year about 500 million diarrhea
from different areas of Kohat and Mohamand
cases take place in children usually less than five
Agency. Around 23 samples were collected from
years in Asia, Africa and Latin America. The
different dug wells, and lakes. The sampling
diseases like typhoid fever, bacillary dysentery,
method was according to standard method given by
infectious hepatitis and other enteric infections are
APHA (2005). Samples were collected aseptically
commonly transferred through contaminated water.
in 250 ml sterilized autoclavable glass bottles in
Apart from this many other diseases are also
the laboratory.
97
Pure Appl. Bio., 2(3): 96-103, Sep- 2013.
Bacteriological Parameters:
viewing gas and keep warm 44.5°C for 24 hours
The experiments for bacteriological analysis of
and observed for gas making.
drinking water were performed at the laboratories
Isolation of E.Coli
of Pakistan council of scientific and industrial
EMB Agar was used for determination of E. coli.
research (PCSIR) complex Peshawar.
All tubes of E. coli broth showing gas were
Total Plate Count:
subcultured by streaking on EMB Agar plates and
Total plate count is determined by standard
incubated at 35°C for 24 hours. Positive plates
method. Serial dilution was prepared and aliquots
contained typical colonies with metallic sheen were
of 1 ml were added to each replica petri dishes.
inoculated on PCA (plate count agar) slants for
Plate count agar was added to every petri dish for
morphological and biochemical tests. After 24
total plate count and incubated at 35°C for 48
hours incubation, the typical colonies were
hours. After incubation, colonies were formed and
confirmed by biochemical tests and also by kits (E.
then counted by colony counter and results were
coli O157:H7 latex test reagent kit Pro Lab.
expressed as CFU/ml.
Canada) (31).
Total Coliform Bacteria:
Results and Discussion
Multiple tube fermentation technique was used to
This study was conducted for the identification of
determine the total coliforms bacteria. One ml from
bacteria in drinking water samples from different
previously prepared serial dilution was incubated
areas of Kohat and Mohamand Agency. In total, 23
in 3 duplicate tubes containing 10 ml of Lactose
samples were collected from the above mentioned
Broth with reversed Durham tubes and kept warm
areas and checked for the presence of bacterial
at 35°C for 24 and 48 hours. Tubes were observed
contamination. The Total Plate Count, Total
for confirmation of gas formation at the end of 24
Coliform, Fecal Coliform, E. coli and Biochemical
hours incubation. Gas making was calculated by
tests were used for the bacterial identification.
gas dislocation in the reversed vial and bubble
Seven out of ten samples collected from Kohat
formation also occurred when the tubes was shaken
were determined to be unfit for drinking due to the
smoothly. Unenthusiastic tubes were reincubated
presence of E. coli as given in Table 1. Three out
for extra 24 hours and observed for a second time
of 13 samples collected from Mohamand Agency
for gas making. A positive tube with gas creation
were identified to be unfit for drinking purposes
and turbidity was subcultured into BGB (Brilliant
(Table 2). The result revealed that 10 (43.5 %) out
Green bile broth). All BGB tubes were kept warm
of 23 samples have microbial contamination and
at 35°C for 48 hours and checked for gas making.
unfit for drinking. The bacteriological analysis
Total Coliform was calculated from MPN table per
revealed that Total Plate Count (TPC) in the
100 ml.
sample collected from Naveed Hotel (867 CFU/ml)
Total Fecal Coliform Bacteria
was higher among all the samples, while that of
Tubes having 10 ml E.C broth with reversed
Kohat Board (162 CFU/ml) was the lowest. The
Durham tubes were kept warm by means of 3 mm
TPC values of all the samples are given in the
loop from the presumptive fermentation tubes
mentioned tables where the samples have the TPC
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Ayaz et al
Table 1: The analysis of water samples collected from different localities of Kohat.
S. No
Sample
ID
Source
Total Plate
Count
(CFU /ml)
Total Coliform
Bacteria
(MPN / 100 ml)
Fecal
coliform
E.coli
Date of
analysis
Date of
report
(2013)
Remarks
1
Kohat Bazar
Tape Water
688
9.2
present
present
25-Feb
01-Mar
Unfit
2
Kohat Adda
Tape Water
543
3.6
Present
Absent
25-Feb
01-Mar
Unfit
3
KUST
Tape Water
90
< 1.1
Absent
Absent
25-Feb
27-Feb
Fit
4
Kohat Board
Tape Water
162
3.6
Present
Absent
04-Mar
08-Mar
Unfit
5
B-1 Hostel
Tape Water
73
< 1.1
Absent
Absent
04-Mar
06-Mar
Fit
6
Kakaji Hotel
Tape Water
451
3.6
Present
Absent
18-Mar
22-Mar
Unfit
7
Pak Afghan
Hotel
Tape Water
595
9.2
present
present
18-Mar
22-Mar
Unfit
8
Naveed Hotel
Tape Water
867
>23
Present
Present
18-Mar
22-Mar
Unfit
9
Kohat Cantt
Tape Water
67
< 1.1
Absent
Absent
18-Mar
20-Mar
Fit
10
Lake Water
Lake Water
604
>23
Present
Present
18-Mar
22-Mar
Unfit
within safety ranges of WHO was considered as fit
pure water for drinking and washing. These hotels
for drinking and vice versa. Based on all the data
are mostly located in the surroundings of the Kohat
and the given limits of WHO, all the water samples
University where mostly the young generation and
were considered as either fit or unfit for the
specifically students are doing their lunch/dinner.
drinking. The results revealed that the samples
This will have long lasting effect on the health of
collected
highly
the local community. The spring water was mostly
contaminated and this could be due to the open
found safe and free of contamination. Mostly these
access of the lake water to all the available wastes
springs are available in the areas have less
of those areas and no proper sanitation. The lake
population and much better environment, however
water contamination is not only alarming for the
when the spring waters comes in the pipelines and
animals using this water for drinking but also for
provided
humans who are using these lakes for swimming in
contaminated. According to previous literature,
the summer season of Pakistan. In this study, the
90% of rural drinking water supplies system
water belonging to different hotels of the
studied were found contaminated with Coliforms
mentioned areas were found highly contaminated
[31, 32]. The methods used in this study were also
and this is a question mark for the local authorities
previously used to investigate the microbial content
to adopt preventive measures and advise the hotels
of different waters in developing countries. Even
administration to improve their sanitation and
some indicators were used to indicate surface
water storage system in order to provide safe and
contamination of drinking water supplies [33-35].
from
lake
water
were
99
to
local
community,
it
becomes
Pure Appl. Bio., 2(3): 96-103, Sep- 2013.
Table 2: The analysis of the water collected from different areas of Mohmand Agency
S.No
Sample
ID
Source
1
Mohamand Bazar
Tape Water
Total Plate
Count
(CFU /ml)
94
Total Coliform
Bacteria
(MPN / 100 ml)
< 1.1
Fecal
coliform
E.coli
Date of
analysis
Date of report
Remarks
Absent
Absent
11-Mar
13-Mar-13
Fit
2
Gandao
Tape Water
488
>23
present
present
11-Mar
15-Mar-13
Unfit
3
Mohamand
Edu.academy
Tape Water
89
< 1.1
Absent
Absent
11-Mar
13-Mar-13
Fit
4
Prang Ghar
Tape Water
47
<1.1
Absent
Absent
11-Mar
13-Mar-13
Fit
5
Spring
Water
Tape Water
267
9.2
Present
Absent
15-Mar
19-Mar-13
Unfit
6
Yaka Ghund
Cheena
Subhan Khawar
91
< 1.1
Absent
Absent
15-Mar
18-Mar-13
Fit
7
Sesai Kelli Khawar
Lake Water
276
9.2
Absent
Absent
23-Apr
26-Apr-13
Unfit
8
Yar Khan Kelli
Tape Water
36
< 1.1
Absent
Absent
23-Apr
25-Apr-13
Fit
9
Khwaga Kelli
Tape Water
64
< 1.1
Absent
Absent
25-Mar
27-Mar-13
Fit
10
Kharei Dara
Tape Water
62
< 1.1
Absent
Absent
15-Apr
18-Apr-13
Fit
11
But Mena Kelli
Tape Water
90
< 1.1
Absent
Absent
22-Apr
24-Apr-13
Fit
12
Pie Khan Kelli
Tape Water
37
< 1.1
Absent
Absent
22-Apr
24-Apr-13
Fit
13
Ambaar
Tape Water
81
<1.1
Absent
Absent
29-Apr
02-Mar-13
Fit
E. coli in drinking water was also used for the
knowledge,
identification of recent fecal contamination and for
placement of wells and the poor ways of water
the identification of pathogens, or intestinal
supply. Mostly these chances were reported in high
parasites [36]. Almost, 15% of the drinking water
ratio during and after rainfall periods [31]. It is
was reported to be contaminated with Coliforms
very important to educate the local community and
instead of up-to-date standards. In case of major
hotel administrations regarding the hazards of the
cities such as Karachi (Pakistan) some pathogens
water pollution and safety measures to prevent
such
microbial contamination in drinking water.
as
Bacillus
Flavobacterium
sp.,
sp.,
preudomonads.,
Actinomyces.,
and
leakage
of
pipelines,
improper
Conclusion
Micrococcus sp were reported in water samples
It can be concluded from the study that the spring
where the Coliform is absent and this revealed that
and dug well waters were mostly pure and free of
some pathogens can even inhibit the Coliform
contamination, however when these waters go
growth and thus results its exclusion from water
through pipelines the water becomes contaminated.
[37, 38]. In this study, the water samples collected
The reason is the unsafe routes and leakage in the
from the springs or dug wells were found safe,
pipelines and should be properly managed to
however when these waters moved in the pipelines
reduce contamination. The lake water was highly
or stored in the containers, the contamination
contaminated as it provides an open space for
chances increased. This could be due to lack of
waste water, industrial solid waste and gutter waste
100
Ayaz et al
Table 3: The bacteriological standards fixed by World Health Organization (WHO).
Bacteriological Parameters
Standards
Total Plate Count
<100 cfu/ml
Total Coliform Bacteria
<3 MPN/ml
Total Fecal Coliform Bacteria
<3 MPN/ml
E. coli
- ve
Table 4: Biochemical test for confirmation of E. coli in waters of Charsada and Mohamand agency.
S. No
Sample ID
MRVP
Indole test
Latex
1.
Kohat Bazar
Positive
Positive
Positive
2.
Kohat Ada
Positive
Positive
Positive
3.
Kohat Board
Positive
Positive
Positive
4.
Kakaji hotel
Positive
Positive
Positive
5.
Pak Afghan hotel
Positive
Positive
Positive
6.
Naveed hotel
Positive
Positive
Positive
7.
Lake water
Positive
Positive
Positive
8.
Gandao
Positive
Positive
Positive
9.
Yaka Ghund Cheena
Positive
Positive
Positive
10.
Cessai Kelli Khwer
Positive
Positive
Positive
of the local population. The water collected from
business community about the hazards of water
hotels was also found contaminated and needs
pollution and the safety measures in order to
proper assessment of the local authorities. Proper
reduce water pollution in the regions.
training is required for the local population and
district, Central Iran. World app. Sci. J., 16: 799-
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