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Determination of Microbial load of Drinking Water from different areas of

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Article  in  Biologia (Lahore, Pakistan) · January 2015

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BIOLOGIA (PAKISTAN) 2015, 61 (1), 151-156
PKISSN 0006 – 3096 (Print) ISSN 2313 – 206X (On-Line)

Determination of Microbial load of Drinking Water from different areas of

Lahore
*UZMA HAMEED1, ABU BAKAR MUHAMMAD1, ARFAA JAHNGEER1 & IKRAM-UL-HAQ1

1
Institute of Industrial Biotechnology, GC University, Katchery Road, Lahore, Pakistan

ABSTRACT
The present study describes the microbial examination and quality assessment of drinking water samples
collected from different localities of Lahore, Pakistan. Microbial load (viable count, spore former, mold and yeast
count) was determined using serial dilution and spread plate method. Bacterial colonies in tap water ranges from
the maximum value of 1.05 x104 to the minimum value of 2.55 x108 CFU/ml, former colonies in tap water, range
from 1.30 x102 to 2.75 x105 CFU/ ml. Water sample collected from Shalamar town contained highest Total
Mean Viable Count CFU/mL, 7.00×105. However, Lahori Gate water sample contained highest number of spore
former, 8.00×102.
Keywords: Microbial load, drinking water contamination, drinking water quality

INTRODUCTION drinking and recreational water (Besser, 1999). Wild

Water covers 71% of the earth surface and
is essential for all the living beings. Almost all of the
biochemical reactions occur in the presence of
water. Drinking safe water has several health
benefits like regulating appetite, increase
metabolism, boost energy levels and help reduce
blood pressure. Water also helps to maintain the
internal body temperature and maintains a constant
fluid balance. Microbes are integral part of water
that are not only responsible for nutrient recycling in
marine and fresh water environment but can also
contribute to variety of water born diseases (Willey
et al., 2008; Ihle et al., 2014; Ravet & Brailowsky,
2014). Most common diseases caused by
contaminated water are diarrhea, dysentery,
pyogenic infections, gastroenteritis, eye, ear and
skin infections, malaria, yellow fever,
shistosomiases, dengue fever, dyspepsia and
urinary tract infections (Bharti et al., 2003).
Many pathogens microbes such as
Spirochete, Rickettsia, Escherichia coli, Shigella,
Salmonella, Enterobacter, Klebsiella, Citrobacter,
Novoviruses, enteric hepatitis viruses,
gastroenteritis viruses, enteroviruses and parasitic
worms i.e. helminthes are present in water (Bharti et
al., 2003; Bosch, 2007). In addition, different kinds
of molds such as Aspergillus spp. Penicillium spp.
are also present in drinking water that are usually
allergic and toxigenic (Hageskal et al., 2006). These
fungi are not only accountable for the adverse
effects on health but also cause taste and odor
problems in drinking water (Dogget, 2000).
Water born E. coli is transmitted by direct
contact with infected farm or pet animals or their
feces (Locking et al., 2001) and from contaminated
and farm animals grazing in water catchments are
sources of faecal contamination of water and water
borne E. coli 0:157 infections (Chalmers et al.,
2000). Molecular techniques have been successfully
used for the detection of pathogens in drinking
water distribution systems (DWDS) especially in
systems with biofilms (Giao, 2011). However,
general laboratory methods are used to determine
the viable count, spore formers, coliform and mold
and yeast number can still effectively be used for
the determination of level of contamination in
drinking water. Multiple Tube Fermentation test
(MTFT) is a standard method that involves three
successive steps, namely, presumptive test,
confirmed test, and completed test, to establish the
presence of coliform. It has been in use for over 80
years as a water quality monitoring method (Edberg
et al., 1988).
The present study describes the microbial
examination of tap water obtained from different
localities of Lahore to find out whether the water
samples from these areas are free from
contamination and safe to drink.
MATERIALS AND METHODS
Sampling
Tap water samples were collected from
different localities of Lahore in sterile polythene
bags.
Viable count
Petri plates of nutrient agar medium
containing (g/L): casein hydrolyzate 4.0, yeast
extract 3.0, glucose 2.0, beef extract 1.5, peptone
6.0 and agar 20.0 (pH 7.0) were used to determine

*Corresponding author: uzmahameed@gmail.com

152 U. HAMEED ET AL
viable bacterial count. 0.5 ml of each sample was
transferred to the individual plates for determination
of total viable counts (TVC) followed by incubation
at 37ºC for 24-48 h. Each sample was run in
triplicates (Anon, 1994). Total number of colonies on
each plate were counted with the help of colony
counter and Colony Forming Unit (CFU) was
counted using formula:
CFU = No. of colonies x Dilution factor /
sample volume (ml)
Spore former
Heat shock treatment was given to the
water samples by keeping them at 90ºC in water
bath for 15 min followed by immediate cooling to
room temperature. Transferred 0.5 ml of each
sample was transferred to the individual Petri plates
containing nutrient agar medium. These plates were
then incubated 37oC for 24 h followed by colony
counting (Anon, 1994).
Mold and Yeast Count
Petri plates containing malt agar medium
(malt extract 3%, mycological peptone 0.5% and
agar 1.5%, pH 5.5) and YPD agar medium (yeast
extract 1%, peptone 2%, dextrose 2%, agar 1.5%
and ampicillin 100 μg/μl, pH 6.5) were used to
determine the presence of mold and yeast,
respectively. The plates containing 0.5 ml of water
samples were incubated at 25-30ºC. After 24-72 h,
the number of mold and yeast colonies was counted
by colony counter (Anon, 1994; Rompre et al.,
2002).
Coliform Detection Test
The coliform detection was carried out by
standard multiple tube fermentation technique.
Presumptive Test: The presumptive test was
carried out using lactose broth medium containing
malt extract (3.0 g/L), peptone (10 g/L), lactose (5.0
g/L) and Bromothymol blue indicator (1.0 g/L).
Transferred 10 ml of lactose broth medium in test
tubes. After that added inverted Durhams tubes and
5 ml of water sample in it. After 24 h incubation at
37ºC the test tubes were examined for the presence
of acid and gas. Acid production is indicated by a
change in color and while gas production is
indicated by the accumulation of gas bubbles in the
inverted Durham tubes.
Confirmed Test: Eosine Methylene Blue (EMB)
agar medium containing peptone (10 g/L),
potassium phosphate (2.0 g/L), Lactose (1.0 g/L),
BIOLOGIA (PAKISTAN)
Eosin Y (2.0 g/L) and Methylene blue (1.3 g/L)and
agar (20 g/L) was used to perform the confirmed
test. A loop full of culture from each positive
fermentation tube was streaked over the sterile agar
EMB plates. The plates were incubated at 37ºC for
24 h.
Complete Test: A final check of colonies that
appear after confirmatory test was performed by
growth on the nutrient agar slant and in Durham
tube containing lactose broth. After incubation for
24h at 35ºC, the lactose broth was examined for the
production of gas. Bacterial cultures from nutrient
agar slants were used to prepare a slide and after
gram staining, it was examined under microscope.
The presence of gram negative, non-spore forming
rod that ferments lactose, confirmed the presence of
coliforms in the water samples (Feng et al., 2002).
RESULTS AND DISCUSSION
Total viable count for each sample was
determined and it was observed that all the samples
showed different ratio of microbial load. Bacterial
colonies in tap water of different areas range from
1.05 x104 to 7.0 x105 CFU/ml, while the bacterial
colonies in the bottled water range from 1.05 x107
to 3.5 x106 CFU/ml. Whereas, maximum viable
count was obtained in Sadar, Garhi shaho and
Lakshami chowk in present work (Table I). Mariam
et al. (2000) reported TVC for tap water of different
areas of Lahore ranged from 20-29 per ml.
Presence of spore former bacteria was determined
by giving heat shock to the water sample at 90ºC for
15 min followed by incubation at 37ºC for 24 to 28 h.
These were in range of 1.25 x103 to 8.0 x102
CFU/ml with maximum spore former count in water
sample of Lahori gate, Singh pura and begum pura
and minimum 1.55 x107in the area of Shad Bagh
(Table I). Thus, results showed that spore formers
are present in almost every water sample except
water sample collected from Lahore hotel and
Anarkali.
The presence of mold and yeast was also
determined in the present work as they can cause
water contamination and serious health hazards, the
detection of mold and yeast in the water samples
collected from the different areas is shown in (Table
II). Mold and yeast were present in areas of
Darogha wala, Singh Pura, Sadar and Wapda
Town. Various fungal frequencies have been
reported from different countries; South Africa
fungal frequencies are 0-3x103 CFU / 100 ml
(Augustionos et al., 1995). In Greece, 36.6 CFU /
100 ml of Tap water samples is detected. These
colonies are of filamentous fungi (Arvanitidou et al.,
2000. According to a previous study in Pakistan the
VOL. 61 (1) DETERMINATION OF MICROBIAL LOAD OF DRINKING WATER 153

majority of the mineral water samples were In the present work coliform detection was
contaminated with P. aeruginosa and E. coli (Taj & carried out using MTFT and (Table I and II) depicts
Baqai, 2007) the results. The samples collected from the areas of
Sadar and China scheme were positive for the
coliform presence.
Table I: Total viable count and spore formers of water samples
collected from different areas of Lahore
Characteristic of
Sample predominant colony Total Mean Viable
Spore former
S. No. Location Count
Color Morphology
(CFU / ml)
(CFU /ml)
1. Anarkali Yellow Cocci 1.05×104±0.071 -
2. Lahori Gate White Bacilli 1.55X106±0.071 8.0X102±0
3. Baghanpura Pale Cocco-bacilli 2.3X106±0 2.05X103±0.071
4. Shalamar Yellow Bacilli 7.0X105±0 -
5. Singh Pura Off white Cocci 1.85X105±0.071 5.05X102±5.587
6. Mehmood Booti Yellow Bacilli 2.365X106±0.049 1.65X104±0.071
7. Darogha wala White Cocci 2.55X105±0.071 1.8X102±0.141
8. Lakxmi Chowk Pale Yellow Cocco-bacilli 2.65X107±0.071 2.45X103±0.751

9. Akbari mandi White Cocci 1.8X106±0.141 1.35X102±0.212

10. Shad Bagh White Bacilli 1.55X107±0.071 1.25X103±0.751
11. Delhi Darwaza White Bacilli 2.8X106±0.141 2.35X103±0.751
12. Sanda Yellow Cocci 2.9X106±0.141 2.2X105±0.141
13. Mozang Off-white Bacilli 2.8X107±0.141 2.4X102±0.424
14. Ichra Yellow Cocci 1.65X107±0.212 1.25X104±0.354
15. Liberty Pale-yellow Bacilli 2.45X108±0.071 1.2X105±0.414

16. Johar Town White Cocci 1.85X106±0.071 1.45X102±0.075

17. Town ship White Bacilli 2.35X106±0.071 2.1X103±0
18. Wapda town Yellow Cocci 3X107±0.141 2.75X103±0.071
19. Garhi Shaho Yellow Cocci 2.55X108±0.071 1.95X104±0.212
20. Bhagat Pura Off-white Cocco-bacilli 2.25X105±0.212 1.9X103±0.141
21. Lahore Hotel Yellow Bacilli 1.5X105±0.707 -
22. Shimla White Cocci 1.25X106±0.071 4.0X104±1.414
23. Green Town Yellow Cocci 1.45X107±0.071 5.5X103±2.121
24. Bhati Gate White Cocci 1.55X106±0.071 1.3X102±0.141
25. Gulberg Off-white Bacilli 2.1X105±0 1.65X103±0.495
154 U. HAMEED ET AL BIOLOGIA (PAKISTAN)

26. Sadar Pale yellow Cocci 2.75X107±0.071 2.05X103±0.071

27. China scheme Yellow Bacilli 2.05X106±0.071 1.5X103±0.707

28. Bata Pur White Bacilli 2.35X108±0.354 1.4X102±0.283
29. Dhobi ghat White Cocci 2.75X107±0.071 1.75X103±0.071
30. Mughal pura Off white cocci 2.35X107±0.071 2.75X105±0.071
31. Begum pura Off white Bacilli 1.3X105±0 7.5X103±0.707
32. Ghoray shah Pale yellow Bacilli 2.45X105±0.071 1.5X102

33. Chah Meeran White Cocco Bacilli 1.1X107 4.0X104

34. Cantt. Yellow Cocci 1.8X107±0 3.5X104±0.707
35. Jalo Off White Bacilli 1.05X106±0.071 2.5X103±0.707

Table II: Presence of fungi and faecal coliform in the water samples collected
from different areas of Lahore
Fungi Detection
Sample Faecal Coliform Detection Test
Test
S. No. Location
Presumptive Confirmed Complete Gram
Mold Yeast
test test test staining
1 Anarkali - - - - -
2 Lahori Gate + - + + + Negative
3 Baghanpura - - - - -
4 Shalamar - - + + + Negative
5 Singh Pura + + - - -
6 Mehmood - - - - -
Booti
7 Darogha wala + - + + + Negative
8 Lakxmi Chowk - - - - -

9 Akbari mandi - - - - -
10 Shad Bagh - - - - -
11 Delhi Darwaza + + + + + Negative
12 Sanda + - + + + Negative
13 Mozang + + + + + Negative
14 Ichra - - - - -
15 Liberty - - - - -

16 Johar Town - - - - -
17 Town ship - - - - -
18 Wapda town - - + + + Negative
VOL. 61 (1) DETERMINATION OF MICROBIAL LOAD OF DRINKING WATER 155

19 Garhi Shaho - - - - -
20 Bhagat Pura - - - - -
21 Lahore Hotel - - - - -
22 Shimla - - - - -
23 Green Town - - - - -
24 Bhati Gate - - - - -
25 Gulberg - - - - -
26 Sadar + - + + + Negative

27 China scheme + - - - -
28 Bata Pur - - - - -
29 Dhobi ghat - - - - -
30 Mughal pura - - - - -
31 Begum pura - - - - -
32 Ghoray shah + + - - -

33 Chah Meeran - - - - -
34 Cantt. - - - - -
35 Jallo - - - - -

CONCLUSION Augustionos, M.T., Venter, S.N. & Kfir, R., 1995.

The study demonstrated that faecal
pollution in natural streams of Lahore has intensified
due to lack of proper municipal waste facilities
particularly in semi-urban areas. In the present
studies tap water samples were collected from
different localities of Lahore to determine the
microbial load. Water of major areas of Lahore has
been found contaminated with faecal coliform and
not viable. Water from Shalimar, Township and
Sadar was found to be positive for the presence of
coliform and is not potable.
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