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Journal of Water Research.

Photon 137 (2015) 295-299


https://sites.google.com/site/photonfoundationorganization/home/journal-of-water-research
Original Research Article. ISJN: 3294-9473: Impact Index: 4.62

Journal of Water Research

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Bacteriological Analysis of Water in a Tertiary Care Center Reveal Coliforms


and Non-coliforms
M. Bhattacharjee, A.D. Urhekar, R. Sharma*
Department of Microbiology, MGM Medical College and Hospital, Kamothe, navi Mumbai Sector 1, Navi Mumbai 410209,
Maharashtra, India
Article history:
Received: 14 September, 2014
Accepted: 17 September, 2014
Available online: 30 December, 2015
Keywords:
Drinking water, Coliform, MPN
Abbreviations:
MPN: most probable number, GNNF: Gram negative non
fermenters
Corresponding Author:
Sharma R.*
Assistant Professor
Email: revupattani ( at ) gmail ( dot ) com
Bhattacharjee M.
Ph.D Student
Urhekar A.D.
Professor & Head

Abstract
Introduction: The microbial quality of drinking water is
of utmost importance and can be defined using
microbiological tools, indicator organisms. These
organisms show the possibility of pollution of water with
pathogenic organism excreted in faeces. The presence of

biological indicators of water pollution like E.coli and


Streptococcus faecalis in drinking water indicates recent
pollution and that of the hardier spore bearing
Clostridium welchii indicates remote pollution.
Experiment: A total of 100 water samples were analysed
over a period of one year by Plate count (Pour plate),
Presumptive coliform count (A quantitative test for all
Coliform Bacilli/100ml) and Differential Coliform test
(A differential test for faecal and non-faecal coli).
Results: Organisms isolated were E.coli (1%),
Citrobacter species (9%) and Non coliforms (16%)
consisting of Gram negative non fermenters (3%),
Pseudomonas species (4%) and Acinetobacter species
(9%). Conclusions: The findings of this study sheds light
upon the importance of routine microbial analysis of
water in hospital setups. The quality of water is even
more crucial when the targeted population is the ailing or
recovering patients.
Citation:
Bhattacharjee M., Urhekar A.D., Sharma R., 2015.
Bacteriological Analysis of Water in a Tertiary Care Center
Reveal Coliforms and Non-coliforms. Journal of Water
Research. Photon 137, 295-299
All Rights Reserved with Photon.
Photon Ignitor: ISJN32949473D749030122015

1. Introduction
Safe drinking water remains inaccessible to several
millions people in the globe. Contamination of
drinking water due to natural and manmade
contaminants is frequently reported in developing
countries, where mainstream of the inhabitants
survive in countryside and uptown areas with
meager
hygiene
and
waste
clearance
practices (Jensen P.K., 2002). Opportunistic
pathogen infections are a serious public health
problem, especially to debilitated persons, in any
areas where large numbers of people are in close
confinement, particularly hospitals and senior care
facilities. At least five percent of patients admitted
to hospitals acquire nosocomial infections and
about one percent of the affected patients die as a
direct result. Many of these organisms are found

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among the diverse flora of water


(Grabinska-Loniewska et al., 2007).

supply

Hospital drinking (potable) water systems is the


most important and controllable, as well as the
most overlooked source, of nosocomial pathogens.
Patient exposure to waterborne microorganisms in
the hospital occurs while showering, bathing,
drinking water, or ingestion ice. It can also occur
through contact with contaminated medical
equipment such as take feed bags, of flexible
endoscope and respiratory equipment that have
been rinsed with tap water. The hands of healthcare
personnel washed using tap water can also lead to
patient exposure to opportunistic pathogens. Our
hospital settings receive treated water from the
municipal services; however, the stagnant water

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sections can be the areas which might be teeming


with the pathogens and can be one of the important
sources of nosocomial infections.
2. Objective of Research
The current study was focused on analyzing the
quality of portable water in the hospital setup.
Routine examination of drinking water in such an
area is of prime importance because the individuals
have higher degree of susceptibility to water born
diseases or nosocomial infections imparted by
water. The results obtained are expected to shed
light on the bacteria present in the portable water of
the hospital and the seriousness of its presence in it.
The presence of coiform as well as certain non
coliform bacteria leads to direct contact of the
patients with them, thereby increasing the chances
of hospital acquired infections.

3. Materials and Method


The study was carried out in the Department of
Microbiology of a tertiary care center in Navi
Mumbai, Maharshtra, India for a period of one
year. Samples of water from different locations in
the hospital campus (n=100) were collected and
subjected to MPN test and Eijkman test for
bacteriological analysis.
3.1. Collection of water samples
Water samples (n=100) were tested from 20
different endpoints (direct taps and water coolers)
in five different months namely April (direct taps
and water coolers) (Summer), June (Early
Monsoon), August (Late Monsoon), October
(Withdrawal), December (Winter). Samples were
taken to simulate every-day usage: with previous
flushing, at usage temperature and without previous
tap disinfection. One hundred-millilitre samples
were collected at a time for the purpose of analysis.

Table 1: Source of water samples in the hospital setting


Sr. No

Location

1
2
3
4
5
6
7
8
9
10
11
12
13

Boys Hostel Water Cooler (WC1)


Girls Hostel Water Cooler (WC 2)
PG Boys Hostel Water Cooler (WC 3)
PG Girls Hostel Water Cooler (WC 4)
Maxillofacial Ward Water Cooler (WC 5)
O.T. Water Cooler (WC 6)
Male Medicine Ward Water Cooler (WC 7)
Ophthalmology Ward Water Cooler (WC 8)
Hospital Mess Water Cooler (WC 9)
Library Water Cooler (WC 10)
1st Floor College Water Cooler (WC 11)
Ground Floor College Water Cooler (WC 12)
Engineering Canteen Water Cooler (WC 13)
Total water cooler samples
14
Boys Hostel Mess (TW1)
15
Girls Hostel Mess (TW2)
16
Maxillofacial Ward (TW3)
17
Casualty Canteen (TW4)
18
Physiotherapy Canteen (TW5)
19
Engineering Canteen (TW6)
20
Microbiology Department (TW7)
Total tap water samples
Total water samples from 20 different locations

3.2. Media and Methods


The media and methods used in this study were in
accordance with the Standard methods for
examination of water. In brief.
Presumptive coliform test:
An estimate of the number of coliform organisms is
usually made by adding varying quantities of water
(0.1 to 50ml) to bile salt lactose peptone water
(with an indicator for acidity) and incubating at
appropriate temperatures. Acid and gas formation
indicate the growth of coliform bacilli.The
probable number of coliforms per 100ml is read off
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No of times samples studied


5
5
5
5
5
5
5
5
5
5
5
5
5
65
5
5
5
5
5
5
5
35
100

from the probability tables of McCarty.


Differential coliform test:
The coliform count does not discriminate between
the Escherichia coli of faecal origin and the non
faecal intermediates. For this differentiation,
subcultures are made from all the bottles showing
acid and gas into fresh tubes of single strength
(5ml) MacConkeys broth and Tryptophan broth
for indole test. Finally incubated at 440C water bath
and examined after 48 hours. Those yielding gas
and showing indole positive were regarded as
containing Escherichia coli and consumption of the

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number in 100ml of water can be made as before.


This is the Confirmed Escherichia coli count.

Heterophilic plate count was performed by spread


plate technique for determining bacterial count.

Characterization and preliminary identification of


the bacteria were made on the basis of morphology
and motility, colony characteristics, biochemical
reactions and carbohydrate fermentation test
conducted as per the standard procedures.

4. Results

Subcultures from the positive presumptive coliform


tests were subjected to Eijkman test for fecal
coliform and confirmed E.coli count by incubating
in a lactose lauryl tryptose broth other subculture at
440C of gas from lactose and indole from
tryptophan.

Of the100 samples studies, 74 did not show


bacterial growth, 26 samples showed positive
bacterial growth as shown in table 2. Organisms
were E.coli (1%), Citrobacter species (9%), Non
coliforms (16%). Of the non coliforms, 3% were
Gram negative non fermenters, 4% were
Pseudomonas species, 9% were Acinetobacter
species

Table 2: Percentage of the various types of organisms isolated form water samples
ORGANISM

PERCENTAGE (%) OF ISOLATES NUMBER OF ISOLATES

Escherichia coli
Citrobacter species
Acinetobacter species
Pseudomonas species
Gram negative non-fermentor
Sterile

1%
9%
9%
4%
3%
74%

1
9
9
4
3
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4.1. Analysis of tap water samples


Table 3: Location of tap water samples, number of samples studied, month-wise isolation of bacteria
No of samples showing
Sr. No. Season.
Samples studied.
No of bacterial isolates.
bacterial growth.
1
1
1.
April (Summer) 7
i) Girls Hostel Mess
i) (Citrobacter spp.)
3
2
June (Early
i)(Citrobacter spp. +
2.
7
i) Girls Hostel Mess
Monsoon)
Acinetobacter spp.)
ii) Casualty Canteen
ii) (GNNF)
6
3
i)(Citrobacter spp.+
August (Late
i) Girls Hostel Mess
Acinetobacter spp.+
3.
7
Monsoon)
ii) Casualty Canteen
Pseudomonas spp.)
iii)EngineeringCanteen
ii) (E.coli +GNNF)
iii) (Citrobacter spp.)
4
2
i) (Citrobacter spp.+
October
4.
7
i) Girls Hostel Mess
Acinetobacter spp.+
(Autumn)
ii)Engineering Canteen
Pseudomonas spp.)
ii) (Citrobacter spp.)
2
December
1
5.
7
i) (Citrobacter spp. +
(Winter)
i) Girls Hostel Mess
Pseudomonas spp.)
Figure 1: E. coli showing Eijkman Test Positive

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Figure 2: MPN Test showing positive


MPN/100ml=19, Grade= Unsatisfactory)

result

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4.2. Analysis of water cooler samples


Table 4: Location of samples from water cooler, number of samples studied, month-wise isolation of bacteria
Samples
No of samples showing bacterial
Sr No. Season.
No of bacterial isolates.
studied.
growth.
April
1
1
1.
7
(Summer)
i) Girls Hostel
i) (Acinetobacter spp.)
3
3
June (Early
i) Girls Hostel
i) (Acinetobacter spp.)
2.
7
Monsoon)
ii) Casualty Canteen
ii) (Citrobacter spp.)
iii) Male Medicine
iii) (GNNF)
3
3
August (Late
i) Girls Hostel
i) (Acinetobacter spp.)
3.
7
Monsoon)
ii) EngineeringCanteen
ii) (Citrobacter spp.)
iii)Ophthalmology
iii) (Acinetobacter spp.)
2
2
October
4.
7
i) Girls Hostel
i) (Acinetobacter spp.)
(Autumn)
ii)Engineering Canteen
ii) (Pseudomonas spp.)
December
1
1
5.
7
(Winter)
i) Girls Hostel
i) (Acinetobacter spp.)

5. Discussion
MPN (Most Probable Number) studies take into
consideration the isolation of Escherichia coli,
Klebsiella species, Enterobacter species and
Citrobacter species as significant in relation to
fitness for drinking purpose. Other organisms are
considered as saprophytic contamination.
In the study on water samples from water taps,
from various sources, it was seen that Boys hostel
mess, Maxillofacial ward, Physiotherapy canteen,
Microbiology department water samples did not
show any bacterial growth any time meaning good
quality of water. In the Engineering canteen
sample, 5 tap water samples were studied.
Organisms isolated were Citrobacter species at
both times with MPN count of greater than 10 per
100 ml of water indicating unsatisfactory for
consumption. Tap water samples were studied from
Casualty canteen water. Sample in month of June
showed growth of GNNF and sample of August
showed growth of E.coli and GNNF. The MPN
count of E. coli isolated was more than 10 per
100ml of water. Thus was proved unsatisfactory for
consumption.
Water samples from water coolers too showed a
similar picture as that of the tap water.
In this study done with100 samples we can observe
that 10% were coliforms, 16% were non coliforms
and the remaining 74% were sterile.
Of the 10% coliforms 9% were Citrobacter species
and 1% was E. coli. These samples indicate that
water at the monsoon time was not suitable for
drinking purpose and proper corrective measures
were advised to the concerned authority.

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In the water samples from Girls hostel, there was


repeated isolation of Acinetobacter in all months,
indicating contamination of water supply. This
finding was informed to site engineer of the
hospital campus for corrective measures.
Jensen et al., 2002 surveyed drinking water of poor
rural communities with water stored in wide and
narrow necked pitchers. They found that direct
hand contact contaminated the water samples. We
also can conclude that direct contact of water in the
source itself might be contaminating the water at
many locations. The leakage or broken outlet of
water may as well allow the user to come in direct
contact with it.
Ghulam Sarwar et al., 2004 studied bacterial loads
and contaminants in drinking water in and around
Peshawar. Out of 224 water samples, 81%
untreated and 19% of treated water samples were
positive for coliforms. E.coli was found in 43.28%
of the samples. The current study also showed that
drinking water which is treated has less chances of
bacterial contamination as compared to the other
portable water in the hospital setup.
Pathak and Gopal, 1994 studied 89 drinking water
samples, from 56 springs, 28 dug wells, 5 streams
in Rajouri district in Jammu and Kashmir. They
found 49.4% water samples were contaminated
with coliform species. We found 10% coliforms in
the water samples which less compared to the
above findings, the reason for this is that portable
water was analyzed in the present study.
The findings were sent to Medical Superintendent
and Site Engineer to find out the reason for
contamination of water and take corrective
measures.

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Water samples from Girls hostel water tap and


water cooler showed consistent contamination. It
was found that there was stagnant water body and
storage reservoir for recycling of sewage water
near the drinking water pipe lines. Necessary
corrective action was taken by the civil
construction department.

Acknowledgement

Other water samples showed contamination in June


and August possibly due to soil contamination
during rainy season. In this situation, additional
safety measures like filtration of water and boiling
of water for drinking purpose were advised.

Grabinska-Loniewska A.G., Wardzynska E., Pajor D.,


Korsak and Boryn K., 2007. Transmission of specific
groups of bacteria through water distribution system. Pol.
J. Microbiol, 56, 129-138.

Other measures taken were cleaning of overhead


water tanks regularly and additional chlorination
procedure for disinfection of drinking water.
Research Highlights
The major observations found are that portable
water in hospital setups can be contaminated by
coliforms and certain non coliforms. The
saprophytic non coliforms like Pseudomonas will
be important when the water is used in the
intensive care units and operation theatres. Regular
monitoring of the water in such areas is of utmost
importance.
Limitations
The water samples from the source i.e the reservoir
and overhead tank could be analysed to check the
bacteriological quality. This would have let us find
if the main supply of water was properly subjected
to purification process from time to time. Also the
frequency of purification and extra measures
whenever necessary could be determined.

We would like to thank the institution for per


mitting to conduct the research work and providing
us the required facility.
References

Jensen P.K., Ensink J.H., Jayasinghe G., Van der


Hoek W., Cairncross S., Dalsgaard A., 2002.
Domestic transmission routes of pathogens: The problem
of in-house contamination of drinking water during
storage in developing countries. Tropical Medicine and
International Health, 7(7), 604-609.
Ghulam S., Khan J., Iqbal R., Afridi A.K., Khan A.,
Sarwar R., 2004. Bacteriological analysis of
drinking water from urban and peri-urban areas of
Peshawar. Journal of Postgraduate Medical Institute,
18(1), 64-69.
Pathak S.P., Gopal K., 1994. Antibiotic resistance and
metal tolerance among coliform species from drinking
water in a hilly area. Journal of Environmental Biology,
15(2), 139-147.
Senior B.W., (Editors) 1996; The Examination of water,
milk, food and air: Mackie & Mc Cartney, Fourteenth
Edition, Churchill Livingstone, 883-921.

Recommendations
Portable water quality is necessary to be monitored
by conducting bacteriological analysis in a routine
manner. This is of even more necessicity in health
care setups. The chances of post operative
infections transmitted by water can be controlled
by this. A simple method of plate count, followed
by bacterial identification can be adopted for this.
Authors Contribution and Competing Interests
1. Minakshi Bhattacharjee- Research worker.
2. Dr. A.D. Urhekar- Guidance and intellectual
inputs.
3. Dr. Revati Sharma- Guidance and results
interpretation.

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