JOURNAL OF MODERN BIOTECHNOLOGY, VOL. 2, NO.

1, pp 1422, January 2013

Copyright 2013, by Madras Institute of Biotechnology. All Right Reserved.
www.thebiotech.org

Research Article

Enumeration and Characterisation of Coliforms from Automated Teller

Machine (ATM) Centers in Urban Areas
Veerappan Saroja, Senthilkumar Kamatchiammal*, Karthikeyan Brinda and Sakkaravarthy Anbazhagi
National Environmental Engineering Research Institute, Chennai Zonal Laboratory, CSIR Madras
complex, Taramani, Chennai -600113, Tamil Nadu, India.
*Correspondence Author e-mail: kamatchi1956@gmail.com
Received 17 December 2012; Revised 28 December 2012; Accepted 31 December 2012

Abstract
An Automated Teller Machine (ATM) is a computerized telecommunications device that provides the customers of a
financial institution with access to financial transactions in a public space without the need for a human clerk or bank teller.
Diversity of people using the ATM centers is increasing day by day. As ATM centers are usually air conditioned, the cold
and damp environment may favour the growth of micro organisms. This study involves the collection of samples by using
cotton swabs from various ATM centers in Chennai. Sixty five samples were analyzed for the presence of predominant
pathogenic bacteria. Among the 65 samples 27 (41.54%) of the samples were positive for the presence of E.coli, 25
(38.46%) for Klebsiella sp, 8 (12.3%) for Shigella sp. and 6 (9.23%) for Vibrio sp and the total microbial load in nutrient
agar ranged from 40 to 1.9 x 105 CFU. The obtained results were confirmed by using Amplification of target genes, Lac z
and Lam b (commonly present in lactose fermenting microbes) was performed by using Lac z and Lam b primers. Also,
BLAST-n analysis of the sequenced PCR product served as additional confirmation for these studies. As an overview this
study proposes the banks to take preventive and corrective actions against spread of infectious diseases through ATMs and
further alert the users of ATM that it is not only money you draw but also, microbes.
Keywords: Automated Teller Machine (ATM); Coliforms; PCR; BLAST
___________________________________________________________________________________________________
INTRODUCTON
Microbes are the oldest forms of life on earth. Some types
have existed for billions of years. Bacteria are one such
type of microorganisms that are ubiquitous in the
environment, capable of growing on any surface including
liquids when the nutritional and environmental factors are
favourable. Although the vast majority of bacteria are
harmless, a few pathogenic strains of bacteria are found to
be causing infectious diseases.
The delicate natural balance of ecological systems is often
modified through human activities that result in organic
pollutants which bring about either abrupt or gradual
changes in the conditions of the natural environment. With

14 JOURNAL OF MODERN BIOTECHNOLOGY

the continuing expansion and proliferation of urban areas,

and the growing threat of population explosion people
become busy in their day to day life having no time to
withdraw money from bank using the traditional means.
Hence they move with the new advancement called the
Automatic Teller Machine (ATM). Today, the widespread
use of electronic technology in healthcare is another source
of contamination.
In recent years, more people are now moving towards
using the ATMs for their banking needs. According to a
survey by Bank net India, 95% people now prefer this
modern channel to traditional mode of banking, almost
60% people use an ATM at least once a week. As we all
know microbes can survive on many surfaces ranging from
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Saroja et al

those in domestic kitchens and hospitals, to commonly

used office equipment such as computer keyboards,
telephones, mobile phones, it is not a surprise that the
Automated teller machine are also one among them. An
ATM is a computerized telecommunications device.
ATMs are known by various other names including
automated transaction machine, automated banking
machine, money machine, bank machine, cash machine,
hole-in-the-wall and Any Time Money (in India) but there
is one more name which people are not aware of ANY
TIME MICROBES, though this name sounds interesting
its a bitter truth that we all need to accept. As ATM
centers are usually air conditioned, the cold and damp
environment favours the growth of a wide range of
microorganisms from pathogens to harmless microbes.
Healthcare-associated infections are a major concern to
clinicians and healthcare consumers. The emergence of
multidrug-resistant organisms and other pathogenic
microorganisms has made treatment of infections from
these organisms more costly and complex. Studies have
demonstrated a variety of reservoirs in the environment
that have served as sources of contamination (Huang et al.,
2006; De Gialluly et al., 2006; McFarland et al., 1989;
Noskin et al., 1995). Convenient hand-held devices and
various other electronic point-of-care systems these are all
new found sources for infection transmission. A steady
influx of computers in patient rooms or computers on
wheels (COWS) that are moved from bedside to bedside
for electronic documentation will be touched by many
caregivers; and, as a result, harmful bacteria has been
found lurking on computer keyboards thus making it easy
for germs to spread to patients and among healthcare
workers Coliform bacteria are the commonly-used
bacterial indicator of sanitary quality of foods and water.
Schultz et al., 2003; studied 100 keyboards in 29 clinical
areas at an inner-city tertiary-care Veterans Affairs
Medical Center. 95 keyboards were positive for
microorganisms. Streptococcus, Clostridium perfingens,
Enterococcus (including harbour vancomycin-resistant
Enlerococcus faecium), Staphylococcus aureus, fungi and
gram-negative organisms were isolated. Noskin et al.,
2005; studied both Computer keyboards and keyboard
covers to determine their ability to harbour vancomycinresistant Enterococcus faecium (VRE), Methicillinresistant
Staphylococcus
aureus
(MRSA)
and
Pseudomonas aeruginosa (PSEA).
15 VOLUME 2 NUMBER 1 JANUARY 2013

Coliforms from automated teller machines

The keyboard and covers harboured MRSA and VRE Ear

longer periods of time than PSEA. With increased contact
with keyboard there was increased recovery of bacteria on
users' hands. Though the computers could hold these
pathogens, the study also found that they could be easily
cleaned. Rutala et al., 2006; studied the degree of
microbial contamination, the efficacy of different
disinfectants, and the cosmetic and functional effects of the
disinfectant on computer keyboard.
In this study, an attempt has been made to identify the
diversity of bacteria, especially Coliforms, present in ATM
centers from in and around Chennai, Tamil Nadu, India.

MATERIALS AND METHODS

Sample collection
Sixty five samples were collected from different ATM
centers in Chennai. Sampling was done 3 times from these
ATM centers on different days. The samples were
collected using sterile cotton swabs. Sterile test tubes with
screw caps were used to transport the cotton swabs. Inside
the ATM room the swab was taken out and rubbed on the
touch screen and buttons. Samples were collected during
peak hours (i.e.) when more number of people would be
using the ATM centers. Hence sampling was done between
9am to 11am and the samples were brought to the
laboratory within 1 hour. In the laboratory, the samples
were preserved by adding 2 ml of Phosphate Buffer
Solution (PBS) to each test tube under sterile conditions
and stored in a refrigerator at 4oC until use.
All the samples were inoculated as (triplicates) on Nutrient
agar, SS (Salmonella Shigella) agar, TCBS (Thiosulphate
Citrate Bile Salt) Agar and M-EC (Media for E.coli) Test
Agar by spread plate method. Samples were then incubated
at 37oC for overnight. Subsequently 500 l of sample was
processed for DNA isolation. After 24 hrs the incubated
plates were observed for growth of bacteria. A single
colony was isolated and sub cultured on Luria Bertani
(LB) broth for further preliminary and biochemical tests.
The colonies isolated from SS Agar, TCBS Agar and MEC Test Agar only were considered for further tests. Gram
staining was followed to find out whether the isolated
colonies belonged to Gram Positive or Gram Negative
bacterium (Pelczar, 1993). Biochemical tests were

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Coliforms from automated teller machines

performed to confirm the pathogens using IMViC test,

Urease test, TSI test and Carbohydrate fermentation test.
Recovery of DNA
Total genomic DNA was extracted from the PBS buffer
directly by the procedure of Del Sel et al., 1989; using this
procedure, 500 l of samples were used for the isolation of
DNA. DNA from bacterial cells was released by alkaline
lysis with Sodium Dodecyl Sulfate (SDS) treatment. The
Cetyl Trimethyl Ammonium bromide (CTAB) was used to
remove proteins and carbohydrates. The DNA was further
purified by using chloroform-isoamyl alcohol (24:1) and
phenol-chloroform-isoamyl alcohol (24:24:2) extractions
followed by precipitation with isopropanol. After
centrifugation at 12,000 x g for 15 min, the pelleted DNA
was washed once with cold 70% alcohol and dried under
vacuum. That pellets were dissolved by 1x TE buffer.This
procedure was able to recover 100 to 150g of purified
genomic DNA from each sample.
Primers used in this study
Common primer for lactose fermenting group of microbes
which are intended to be coliforms including E.coli, the
marker for faecal contamination, were used for this study.
Bacterial pathogens Primers were chosen to amplify a 326base- pair (bp) region of the LacZ gene, based on the
sequence reported by Kalnins et al., 1983; And also a 554
bp region of the lam B gene based on the sequence
reported by Bedouelle et al., 1980; The primers used in
this study are given in (Table 1).
Polymerase chain reaction
Amplification of a segment of the coding region of
Escherichia coli lacZ by using a PCR primer annealing
temperature of 50C detected E. coli and other coliform
bacteria (including Shigella sp. but not Salmonella sp.) and
non-coliform bacteria. Amplification of a region of E. coli
lamB by using a primer annealing temperature of 50C
selectively detected E. coli and Salmonella and Shigella sp.
Amplification of the DNA extracted from the ATM
samples was carried out as per Asim et al., 1990. PCR
amplification of lacZ and lamB provides a basis for a
method to detect indicators of fecal contamination, and
amplification of lamB in particular permits detection of E.
coli and enteric pathogens (Salmonella and Shigella sp).

16 JOURNAL OF MODERN BIOTECHNOLOGY

Saroja et al

DNA amplification was carried out in gene amplification

PCR system, using 2 X PCR master mixes (Genei). The
PCR master mix normally contained lx PCR amplification
buffer (10x buffer contains 50 mM KCl, 100 mM Tris
hydrochloride [pH 8.13], 15 mM MgCl2, and 0.1%
[wt/vol] gelatin), 200 p.M each of the dNTPs, 0.4 M of
each of the primers and 2.5 U of Taq DNA polymerase
were added to the master mix. Oligonucleotide primers
were synthesized by Integrated DNA Technologies, USA.
After adding the template DNA, the whole mixture was
initially denatured at 94C for 3 minutes. Then a total
number of 30 PCR cycles were run under the following
conditions. Denaturation at 94C for 1 min, primer
annealing at 50C for 1 min, DNA extension at 72 C for 2
min. 10 l of amplified PCR product was electrophoresed
on a 1% agarose gel and photographed under UV light.
DNA sequence
DNA sequencing was carried out using ABI 310 Genetic
Analyser (Shankra Nethralaya, Chennai, India) using both
the forward and reverse primers to confirm the results. The
multiple sequence alignment was analyzed by using
(http://www.align.genome.jp/).

RESULTS
Nutrient agar showed growth of microbes in all the 65
samples ranging from 40 CFU/ml to 9.7104CFU/ml.
According to these results, the highest bacterial
contamination was reported in sample number 32 and the
lowest bacterial contamination was reported in sample
number 2 (Table 2). These samples were further tested
with MEC agar which revealed that 27 out of 67 samples
showed yellow colonies indicating the presence of E.coli.
The result for MEC agar ranged from 20 CFU/ml to
7.4104CFU/ml. The highest number of CFU was reported
in sample number 30. In SS agar Klebsiella sp., 25 samples
tested positive for Klebsiella sp ranging from 80 CFU/ml
to 9.8104CFU/ml. The highest was reported sample
number 26. Then 8 samples indicated the presence of
Shigella sp in SS agar Shigella sp ranging from 40CFU/ml
to 1.7104CFU/ml, reporting the highest in sample number
28. Finally, 6 samples showed positive results in TCBS
agar ranging from 1.2 x 102 CFU to 4.4102 CFU
indicating presence of yellow colour colonies that shows

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Saroja et al

Coliforms from automated teller machines

the existence of Vibrio sp with highest record on sample

number 16. Sample number 30 and sample number 31
showed the presence of all four enteric pathogens. And,
three enteric pathogens namely E.coli, Klebsiella sp,
Shigella sp was found in sample numbers 48 and 50 except
for Vibrio sp. (Table 2). Thus as an overview amongst 65
samples 27 (41.54%), samples were positive for the
presence of E.coli, 25 (38.46%) for Klebsiella sp, 8
(12.3%) for Shigella sp. and 6 (9.23%) for Vibrio sp.
(Figure 1). All samples which showed bacterial growth on
selective media were then confirmed for the presence of

enteric pathogens like E.coli, Klebsiella sp, Shigella sp and

Vibrio sp using cultural, staining and biochemical
examinations. Of the 65 samples analysed, 24 samples
showed growth only on nutrient agar while there was no
growth on the other specific media (i.e.) nearly 36.92% of
the ATM centres are free from enteric pathogens. About
41 samples had one or more enteric pathogens present
(i.e.) nearly 63.08% of the ATM centres possess
potentially enteric pathogens. All the above values
determined are on an average of triplicates.

Table 1: Target Gene and their respective sequences and positions.

Target gene Primers

Sequence (53)

Sense primer

Position

ATG AAA GCT GGC TAC AGG AAG GCC 1675 and 1698

LacZ
Antisense primer GGT TTA TGC AGC AAC GAG ACG TCA 2001 and 2025
Sense primer

GGA TAT TTC TGG TCC TGG TGC CGG

4899 and 4922

Antisense primer ACT TGG TGC CGT TGT CGT TAT CCC

5429 and 5452

LamB

Table2: Expression of bacterial counts in nutrient agar and specific media, MEC agar- E. coli, SS agar Klebsiella sp.,
SS agar Shigella sp. and TCBS agar Vibrio sp. in terms of CFU.

Sample No.

Nutrient agar

Mec agar
E.coli

SS agar-Klebsiella sp.

SS agar-Shigella sp.

TCBS agar-Vibrio sp.

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

4104
40
1.2104
7.2103
5.6103
3.6104
7.2104
1.8104
2.3104
2.6102
5.2104
7104

7.5103
NIL
5.4102
NIL
NIL
NIL
20
NIL
7.6103
NIL
3.2104
40

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
7.2103

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL

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Coliforms from automated teller machines

Saroja et al

13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

2.7104
1.3104
1.6104
6.4103
8.1103
6.5103
8.4104
8.8103
1.1104
1.8105
1.3104
7.8104
8.4104
1.9105
5.7104
4.8104
1.8105
7.8104
1.9104
9.7104
5.2102
2.5103
7.6102
8.4102
3104

1.5103
NIL
1.4104
NIL
NIL
7102
NIL
NIL
60
NIL
2.8103
NIL
NIL
NIL
NIL
3.6104
NIL
3.2104
40
6.4104
NIL
1103
NIL

NIL
7.9103
NIL
4.9104
7.9103
NIL
6.2104
NIL
9.4102
7104
NIL
NIL
8.8103
9.8104
6.5103
NIL
NIL
7.4104
1.5104
NIL
NIL
NIL
NIL

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
1.7104
1.1103
3.2102
2.7104
NIL
NIL
3.1103
NIL

NIL
NIL
NIL
4.4102
3.8102

NIL
NIL

NIL
NIL

NIL
NIL

NIL
NIL

38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53

2102
1.4103
3.4104
4.7103
1.5104
1.4102
2.6102
5.8104
8103
1.1104
4.8104
1.2104
1.6104
7.1104
1.2104
1.9104

NIL
NIL
NIL
NIL
80
NIL
NIL
NIL
1102
8.6102
1.3103
60
4.8104
NIL
NIL
NIL

NIL
NIL
1.2104
NIL
2.9103
NIL
NIL
4.2104
NIL
NIL
5.7104
NIL
5.6103
1.2103
NIL
NIL

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
1.4104
NIL
2.9103
NIL
NIL
NIL

NIL
NIL
1.4102
NIL
NIL
NIL
NIL
3.6102
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL

18 JOURNAL OF MODERN BIOTECHNOLOGY

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
2.9104
60
1.2102
NIL
NIL
NIL
NIL

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Coliforms from automated teller machines

2.7103
1.3104
7.8103
7.1104
5.2104
6102
2.8102
2.6104
1.6102
8.6102
3.9103
5.4103

ATM Samples

54
55
56
57
58
59
60
61
62
63
64
65

70
60
50
40
30
20
10
0

1.2102
NIL
NIL
7.8102
NIL
NIL
1.4102
NIL
60
NIL
8102
NIL

7.2102
NIL
NIL
80
5.1104
NIL
2.4103
1.1104
2.8102
NIL
NIL
NIL

NIL
NIL
NIL
NIL
2.8102
NIL
NIL
NIL
NIL
NIL
NIL
NIL

NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL

Nutirent Agar

MEC Agar

Un known
Microbes

E.coli

SS Agar

Klebsiella sp

SS Agar

TCBS Agar

Shigella sp

vibrio sp

Bacterial Colonies
Figure 1:

Graphical representation on number of coliforms present in ATM sample.

PCR was evaluated for detection of coliforms from 65

samples collected at various ATM centres in Chennai. In
all the PCR positive samples, a distinct 326 bp (lac Z), 554
bp (lam B) products were visible on the agarose gel when
stained with ethidium bromide (Figures 2 & 3). The
identities of the bands were further confirmed by
sequencing. BLAST comparison of the nucleotide
sequence with other sequences available at the GenBank
showed that the PCR amplified sequence comprised of the
LacZ and LamB gene. The accession numbers for the
submitted sequence is HM449754 and HM449759
respectively.

19 VOLUME 2 NUMBER 1 JANUARY 2013

DISCUSSION
In this study an attempt was made to identify probable
diversity of Enteric pathogens that are present in ATM
centers in Chennai, and the results thus identified provides
supporting evidences for pathogenic microbes like E.coli,
Klebsiella spp, Shigella spp, Vibrio spp, to be present at
the ATM centers. The moisture content inside the ATM
centers facilitates the survival of pathogens as the ATM
centers are air-conditioned. This study was conducted to
create awareness to public and scientific community on the
possible diseases that may spread due to the presence of

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Coliforms from automated teller machines

these microbes at the ATM centers. Human occupational

activities, without hygienic intervention, could introduce

Saroja et al

the risk of infection through ATM machines.

Figure 2: Electrophoresis gel results confirming the presence of Lac z regions (326bp) for the samples obtained from
ATMs.
Lane 1: 100 bp Marker
Lane 2: Negative control
Lane 3: Positive control and
Lane 4, 5, 6, 8, 10 ,11, 12, 13, 14, 16, 17, 18 confirming the positive results.
Lane 7, 9 and 15 confirming the negative results.

Figure 3: Electrophoresis gel results confirming the presence of Lam B regions (554bp) for the samples obtained from
ATMs.
Lane 1: 100 bp Marker
Lane 2: Negative control
Lane 3: Positive control and

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Coliforms from automated teller machines

Lanes 4, 5, 6, 8, 10 ,11, 12, 13, 14, 16, 17, 18 confirming the positive results.
Lanes 7, 9 and 15 confirming the negative results.
In this study more focus was given to enteric pathogens, as
coliform bacterias in water using polymerase chain
they are the common ones that spread diseases through
reaction (PCR) amplification and gene probe detection
touch, improper sanitary activities of the individual.
provided as a cutting edge to this research in identifying
Transient pathogens are excreted in faecal, through various
the existence of coliforms in ATM centers. Use of nucleic
body fluids or tissues by persons infected or colonized by
acid techniques provides a rapid identification and
these pathogens (Barker et al., 2005). When diarrhoea
confirmation. Further confirmation with the sequences
results in many liquid stools per day, hands easily become
showed the identities with the LacZ genome in blastn
contaminated because billions of pathogen cells are
analysis.
present. Previous studies also report the presence of
This research is considered to be reliable as the presence of
coliforms in coins, paper currencies, and public telephones
coliforms was detected using consistent methods used by
and the introduction and spread of contamination in those
researchers. This also further provides evidence that even
cases also through handling like in our study (Ferdinandus
ATM machines are risk to handle with, as diseases may
et al., 2001). This work is considered to be important as
spread through the use of these unclean machines. The risk
virulent strains of pathogenic microbes like E.coli,
of food-borne infection associated with crossKlebsiella, Shigella, and Vibrio can cause diseases like
contamination depends on two factors: the level of
gastroenteritis, urinary tract infections, septicemia,
contamination on the surfaces and the probability of its
dysentery, vomiting, stomach cramps, flatulence etc.,
transfer to the foods being consumed (Bloomfield et al.,
Irrespective of all enteric pathogens positive in special
1997). Hand washing before and after use of ATM centers
media were positive for E.coli, the indicator for faecal
may be impractical and imperfect, but it might be desirable
contamination, further confirms the source of
instead to clean the keys and screens with alcohol or other
contamination is due to the insanitary human activities. In
disinfectants on a regular basis. Similar recommendations
this study two ATM centers one from a highly populated
have been made by previous researchers (Neeley et al.,
area and the other near sewage outfalls tested positive for
2005, Rutala et al., 2006 ) and may be pertinent to other
three pathogens, excluding Salmonella. And, four out of
settings, such as the one investigated in this study. This
six samples that reported positive for Vibrio were sampled
research shall serves as a base for researches to identify
from ATM centers nearer to fishermen colonies,
specific strains of species causing disease and effective
supporting the spread of contamination fishes and through
disinfecting procedures that reduce the risk of ATM
handling of marine water. This is supported by the work of
acquired infection.
Khin et al., 1989; where currency notes handled by fish
mongers had contaminations. Even though 22 samples
failed to have E.coli, Salmonella, Shigella and Vibrio they
CONCLUSION
tested positive on nutrient agar indicating the possibilities
Gone are those days where people need to worry while
of microbial contamination other than feacal
carrying cash while travelling to places. Now days in this
contamination. Earlier researches on electronic devices
speedy world ATMs have become a necessary evil. People
like keyboards provide evidences for existence of
often tend to realise that the ATMs not only dispense cash
Streptococcus, Clostridium perfingens, Enterococcus
but also microbes. As the number of ATMs increase day
supports the contamination of ATM surfaces. Also we
by day the increase of pathogens and spread of diseases
observed some rhizoidal colonies on nutrient agar which
tend to increase. Banks have taken high security measures
represents the member of Actinomycetes group indicate
to prevent theft of cash at the ATMs, however they have
the contamination from the soil as they were the common
failed to take preventive and security initiatives on
residents of soil microflora. This research is an extension
maintaining safe and clean ATM machines. Currently, the
to identify pathogenic microbes at ATM machines that is
cleaning of ATMs has reduced drastically and thus more
widely used among all ages of human race (Bej et al.,
contaminated ATMs are present in and around the city. In
1990) on identification of LacZ and lamB regions for

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Coliforms from automated teller machines

this high populated world care should be taken to protect

the human race from spread of infectious diseases for the
betterment of human life.

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