coli-form group and gram Negative bacteria

1. ISSN: 2278-778X
Research Article
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International Journal of Bioassays (IJB) 379
A NEW MEDIUM FOR SCREENING OF GRAM NEGATIVE BACTERIA IN PORTABLE WATER
Mohamed Younes Abdelftah Hassan1
, Omar Abdelateef Omar Saad2
, Magda Hassan Mahran3
, Ahmed Ismail
Madkour4
, Suliman S Abdelrawaf5
and Idress Hamad Attitalla*5,6
1
Minia Company for Drinking Water and Sanitation, Minia, Egypt
2
Department of Agricultural Microbiology, Faculty of Agriculture, Minia University, Egypt.
3
Assalam International Hospital, Egypt
4
Ophthalmology Research Institute, Egypt
5
Department of Microbiology, Faculty of Science, Omar Al-Mukhatr University, Box 919, Al-Bayda, Libya
LibyaAgriculture Research Centre (ARC),6
*Corresponding Author: Dr. Idress Hamad Attitalla, Professor, Department of Microbiology, Faculty of Science,
Omar Al-Mukhatr University, Box 919, Al-Bayda, Libya
Received for publication: October 22, 2012; Accepted: January 03, 2013.
Abstract: A series of laboratory experiments were performed to screen the presence of gram negative
bacteria (coliform group) in drinking water to determine its suitability for human consumption and in
clinical, pathological, and ophthalmology purpose. A new medium (Makka medium) with methylene blue
as an indicator which inhibit Gram positive bacteria was developed, evaluated and used in the present
study. Composition of this medium doesn’t contain lactose found as in m-Endo agar medium. Generally
lactose is used by the bacteria during fermentation process but in Makka medium fermentation occurred
in the absence of lactose which was recorded as the change in the color of the medium due to pH drift
and Dipotassium phosphate dibasic serves as buffer and enhance organism’s growth (coliform group). In
comparison with the commercially available synthetic media, Makka medium is unique in composition,
economical, safe and easy to use for detection of contamination of drinking water with coliforms.
Keywords: Coliform, Gram Negative Bacteria, Makka Medium, M-Endo Agar, Portable Water
INTRODUCTION
Coliforms such as Escherichia coli, E. aurescens, E.
freundii, E. intermedia; Aerobacter, Aerogenes and A.
cloacae are aerobes and facultative anaerobic which
are classified as Gram-negative, non-spore-forming, rod
shaped bacteria which ferment lactose and produce
gas within 48 hours at 35°C. This group has been used
as indicator organisms in most of the water testing
laboratories. An indicator organism, by itself is
considered to cause no diseases in man or animals, but
its presence usually indicates the presence of
pathogenic or disease-causing organisms. 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. However these
methods have their own limitations, such as duration
of incubation, antagonistic organism interference, lack
of specificity and poor detection of slow growing or
viable but non-culturable (VBNC) microorganisms.
Nowadays, the simple and inexpensive membrane filter
technique is the most widely used method for routine
enumeration of coliforms in drinking water. The
membrane filter technique (MF) is used for the
counting of coliforms. The filtration process is designed
in such a way as to retain coliform bacteria present in
the sample onto a membrane filter. The entire filter
with the retained bacteria is placed on a specially
prepared medium in a Petri dish. The Petri dish and its
content are then incubated for 24 hours at 35°C. At the
end of the incubation period, the filter is examined
with a 10-15X stereoscopic optical device. All blue
colored colonies on the filter will then be counted and
each blue colored colony is assumed to be the result of
one faecal coliform originally in the sample. From the
colony counting procedure and the sample volume
filtered, it is possible to calculate the number of faecal
coliforms/ 100 mL present in the sample to be tested
(Mc fetrsetal, 1986; Apha, 1998).
Eosin Methylene Blue (EMB, also known as
"Levine's formulation") is used for testing clinical
materials, food, and dairy products; it is a blend of two
stains, eosin and methylene blue in the ratio of 6:1. A
common application of this stain is in the preparation
of EMB agar, a differential microbiological medium,
which slightly inhibits the growth of Gram-positive
bacteria and provides a color indicator distinguishing
between organisms that ferment lactose (e.g., E. coli")
and those that do not (e.g., Salmonella, Shigella).
Organisms that ferment lactose display “nucleated
colonies" colonies with dark center (Differential Media
(Levine's Formulation)) and E. coli will give distinctive
metallic green sheen (due to the metachromatic
properties of the dyes.

2. Mohamed Younes Abdelftah Hassan et al.: International Journal of Bioassays, 2013, 02 (02), 379-381
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This medium is important in medical laboratories
for distinguishing pathogenic microbes in a short
period of time (Bachoon et al., 2008) (Cunnif, 1995;
Murtay et al, 1995). This medium is primarily used for
detection and confirmation of coliforms. In the
membrane filter technique, them-Endo Agar medium is
usually used for detection of coliforms. In addition to
the high cost of this medium it is reported that this
medium may cause cancer if used without precautions
(Enne de Boer (2004)). Furthermore, some
components in this medium irritate eyes, skin, and
respiratory system. Hence, developing an economical
safe alternative medium for such purpose is a
challenge.
The present study aims at finding a new
economical and safer method for detection of coliform
group in portable water.
MATERIALS AND METHODS
High levels of environmental contamination, often
associated with improper waste and excreta
management, are widespread among informal
settlements within urban areas in developing countries.
We determined the level of fecal contamination in
domestic water sources collected from parts of Egypt
and Libya, evaluated the potential contribution of
these water sources to coliforms using an economical
and indigenous media (Makka media) developed in our
lab. Membrane filtration technique was used for
enumeration of total and fecal (Escherichia coli)
coliform bacteria in water samples collected from
different municipal sources, which is used for drinking
and clinical purposes. A comparative study was
conducted to check if the Makka media developed is on
par with the existing commercially media (m-Endo
Agar) for the detection of Gram negatives especially
coliforms.
i. Sample Collection: The samples chosen for the
study were randomly selected from different
locations across Libya and Egypt during a 2-year
period and checked for routine total coliform
analysis. All samples were collected by accepted
procedures Clinical and Laboratory Standards
Institute guidelines (CLSI)) and shipped to the
laboratory at ambient temperatures. Samples from
municipal water supplies were tested by the
membrane filter total coliform technique.
ii. Isolation of the Microbes
iii. Biochemical characterization
iv. Growth on Makkah medium and m-Endo Agar
v. Colony characteristics
RESULTS
On Makka medium the coliforms appear blue
green color and some have dark green color (Figure
1and4). But in m-Endo agar medium, coliforms form
pink to red colonies and some with metallic sheen were
observed (Figure 5). The first medium is selective in
nature due to the presence of methylene blue, which
inhibits the growth of Gram +ve bacteria but allowing
the growth of Gram –ve ones. It is different in that
lactose fermenting bacteria give colored colonies due
to the formation of complex in this method.
Differential media or indicator media distinguish one
microorganism type from another growing on the
same media Washington (1996). This type of media
uses the biochemical characteristics of a
microorganism growing in the presence of specific
nutrients as indicators (such as neutral red, phenol red,
eosin y, or methylene blue) which are added to the
medium to visibly indicate the defining characteristics
of a microorganism. This type of media is used for the
detection of microorganisms and by molecular
biologists to detect recombinant strains of bacteria.
DISCUSSION
In the 1stexperiment we used Endo agar medium
to confirm the presence of coliform group in the
sample. The coliform group appears with metallic
sheen. This result agrees with Enne de Boer (2004)
who reported that, E. coli also has a characteristic color
(red with green metallic sheen) on Endo agar.
In the 2ndexperiment we used methylene blue and
applied on this medium to see the effect on colony
numbers and characteristics. The coliform group
appear with dark green to pale green due to
consumption of methylene blue.
In the 3rdexperiment we compare the two media
through streaking on filter disc. Data shown in Figs.2
and 3 indicated that, the growth of the coliform group
was identical on both media in the 4th
experiment we
compare both the media through spread culture
technique. These results (figures 6 and 7) showed that,
the efficiency of new medium (Makka medium)
enhance coliform group growth as Endo’s agar
medium. Finally we cultivated typical microorganisms
and compared biochemical tests to determine the
ability of Makka medium towards its selection of gram
negative bacteria.
Generally there is no need to sterilize media for
coliforms. Pasteurization of these media results in
sufficient decontamination, as surviving spore-forming
bacteria will not be able to develop in media with bile
salts and dyes. Moreover sterilization may inactivate
some of the inhibiting substances, making the media
less selective Enne de Boer (2004).
These results showed that Makka medium is
recommended to be used instead of M-Endo agar
medium for detection of coliforms and gram negative

3. Mohamed Younes Abdelftah Hassan et al.: International Journal of Bioassays, 2013, 02 (02), 379-381
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bacteria. To conclude we demonstrated that Makka
medium is equally good in comparison with Eosin
methylene blue agar, Levine used for the isolation and
differentiation of Gram-negative enteric bacilli Madigan
and Martnko (2005), however makka medium showed
the following benefits over EMB agar.
An easy method
Its components' are locally available which are safe and
economical.
It is also guarantees occupational health and safety.
It is no toxic hence can be considered a best alternative
for EMB agar.
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Source of support: Nil
Conflict of interest: None Declared