AIM: Determination of Coliforms in water samples using Eosin Methylene Blue (EMB)
medium.
REQUIREMENTS:
Reagents EMB Agar, Ethanol.
Glasswares Conical Flasks, Measuring Cylinder, Petri Plates, Spreader.
Instruments Autoclave, Laminar Cabinet, Weighing Balance, pH meter.
Others Burner, Gloves, Spatula, Micropipettes, Micro tips.
Water Samples 3-4 water samples from different sources [eg. Tap water, RO water,
pond water, drain water, etc.]
THEORY:
Coliform bacteria- The term coliform was coined to describe the group of enteric bacteria.
Coliform term has originated from Latin word coli & English word form that translates to
bacteria found in the colon. Coliform bacteria are defined as rod shaped, gram negative, non-
spore forming and motile or non-motile bacteria which can ferment lactose with the
production of acid and gas when incubated at 35-37°C. Coliform members include
Escherichia, Citrobacter, Enterobacter, Klebsiella. They are present in intestines and feces of
all warm blooded animals and humans. Coliform bacteria are indicator organisms for
contaminated water with pathogens. Presence of E. coli in food or water is an indicative of
fecal contamination. When cultured on an EMB plate, a positive result for E. coli is metallic
green colonies.
Enterobacter represents group of rod shaped bacteria of the family Enterobacteriaceae. Gram
negative and facultative anaerobes. Enterobacter is named for the organism’s predominant
natural habitat i.e. the intestines of animals (from Greek enteron, meaning “intestine”).
Eosin Methylene Blue (EMB) agar is both selective and
differential culture medium. It is a selective culture medium for
Gram-negative bacteria (selects against Gram-positive bacteria)
& is commonly used for the isolation and differentiation of
coliforms and fecal coliforms. EMB media assists in visual
distinction Escherichia coli, other nonpathogenic lactose-
fermenting enteric gram-negative rods, and the Salmonella and
Shigella genera.
Principle of using EMB media for detection of Coliform
bacteria- Differentiation between these gram-negative
Metallic
bacilli is based on the colony color e.g. colored colonies in green
colonies
EMB agar indicates bacteria is a lactose fermenter while
colorless colonies in EMB agar: indicates that it is a non-
lactose fermenter.
The aniline dyes (eosin and methylene blue) inhibit Gram-
positive bacteria. Hence, EMB is a selective media- They
combine to form a precipitate at acid pH, thus also serving as indicators of acid production.
Gram-negative bacteria that ferment the lactose produce acid which turns the colonies dark
purple as the acid acts upon the dyes. In addition, certain lactose-fermenting bacteria produce
flat, dark colonies with a green metallic sheen. Other lactose fermenters produce larger,
mucoid colonies, often purple only in their center.
In EMB agar, most of the strains of E. coli colonies have a characteristic green metallic
sheen. Rapid fermentation of lactose & production of strong acids, thus a rapid reduction in
the pH of the EMB agar the critical factor in the formation of green metallic sheen observed
with E. coli, rapid fermentation of lactose and formation of strong acids. Lactose non-
fermenters are either colorless or light lavender.
EMB Agar is both an example of differential and selective media
Selective: EMB agar is selective for gram-negative bacteria. The dye methylene blue in the
medium inhibits the growth of gram-positive bacteria.
Differential: EMB agar differentiates between gram negative lactose fermenters that produce
acidic products (E. coli.) and from those that ferment lactose with weak acidic products
(Enterobacter aerogenes and the lactose non- fermenters (Salmonella and Shigella spp.)
Eosin which is an acidic dye reacts with the basic stain methylene blue to form a compound
of either acidic or neutral nature.
o Methylene blue is a synthetic basic dye. It stains to negatively charged cell
components.
o Eosin is a dye that responds to changes in pH, going from colourless to black under
acidic conditions.
The acid produced by lactose fermenters is sufficient to let this dye compound to be taken by
the cells and to make colonies appear dark purple in color. Non lactose fermenters are
colorless because the dye compound cannot be taken up by the cells.
1. Strong fermenters: Lactose-fermenting gram-negative bacteria (generally enteric)
acidify the medium, and under acidic conditions the dyes produce a dark purple
complex which is usually associated with a green metallic sheen. This metallic green
sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of
fecal coliforms.
2. Weak fermenters: A smaller amount of acid production, which is a result of slow
fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration
of growth.
3. Non-fermeters: Colonies of non-lactose fermenters appear as translucent or
colourless.
PROCEDURE:
Preparation EMB Agar [250 ml]:
1. All the components are dissolved in 250 ml D.W. in a flask [read instruction in
package].
2. Swirl the mixture to dissolve any lumps if present.
3. The medium is autoclaved at 121°C for 15 min at 15 psi.
4. Sterilize the workspace before transferring your materials. Light the Bunsen burner.
Allow EMB media to cool before pouring.
5. Take an empty plate and open it slightly [No need to open it all the way to pour agar].
6. Pour agar until 2/3 of the plate is covered, or approximately half the plate has been
filled.
o Pour the agar slowly to prevent bubbles formation. Swirl the plate in a circular
motion to distribute the media evenly.
7. Set the plates to cool in stacks of 4-5 and flip the plates to prevent condensation
forming on agar. Allow to cool for until the agar has solidified.
Inoculation of the sample:
1. In a laminar flow cabinet, Add 10-100 µL of water sample on an EMB agar plate.
2. Spread the culture all over the plate using a sterile glass spreader.
o Or, inoculate the sample by streaking method using a sterile loop/micropipette
tip.
3. Invert and incubate the plates overnight at 37°C.
4. Observe the growth of bacterial colonies and colourization after overnight incubation.
RESULTS & OBSERVATIONS [ELABORATE YOURSELF]
PASTE IMAGES OF THE CULTURE OBTAINED:
In the plates on which sewage sample is plated, there is likelihood of colonies with
green sheen characteristic of E. coli. being observed, along with other bacteria such as
Enterobacter aerogenes and any potable (drinking) water sample, there should be an absolute
absence of colonies typical of E. coli.
PRECAUTION [Write major precautions]:
1. All glass wares should be clean, dust free and sterilized.
2. Always sterilize working area with alcohol before start. Rub your hand with ethanol
before handling the apparatus and culture.
3. Proper care should be taken while working with burner lit in laminar cabinet [ethanol
is highly flammable].
4. Proper care should be taken while spreading or streaking so as not to rupture the agar
plate.
5. Label all plates properly.
