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Also commonly known as "indicator
organisms", coliform refers to a wide variety of bacteria that can be
found throughout the environment. This means that these organisms can be found
in soil, water surfaces, vegetations as well as on the skin or intestinal tract
of warm-blooded organisms such as humans.
Although some are pathogenic (capable of causing diseases - mild to life threatening
diseases) most of them are harmless. Regardless, detection of coliform
(indicator organisms) indicates the presence of potential disease causing
bacteria not only in water, but also in given foods and drinks (milk etc).
Therefore, coliform are important because they help raise awareness and
determine the source of the bacteria.
This group is largely composed of
harmless, closely related bacteria. Apart from human and animal waste, total
coliform bacteria can be found in such environments as water, vegetation and
soil where they live freely.
While they are generally harmless, the presence or
detection of this group of bacteria in drinking water or water source that
supply drinking water is important because they are indicative of possible
If detected in a water sample, this suggests that disease causing
coliform may also be present and thus the need to treat the water source or
determine the source of contamination (environmental contamination etc).
Thermotolerant coliform are good examples of total coliform bacteria. These are
coliform that are capable of fermenting lactose at 45 degrees.
* Detection of total coliform does not
necessarily mean that disease causing bacteria are present in water.
* Testing the presence of total coliform bacteria
basically involves growing them in lactose media at about 35 degrees.
Fecal Coliform Bacteria
Fecal coliform bacteria (FC) are a subgroup of
the total coliform bacteria that can be found in the intestines and feces of
warm blooded animals (human beings, pigs, cows, dogs, pigs etc). E. coli is an
example that typically resides in the intestinal tract of
warm-blooded animals and thus the animal's fecal matter.
When they are outside
the host's body, these organisms cannot live for long because their survival is
largely dependent on the host.
Compared to total coliform bacteria, which are
largely harmless, the fecal are composed of both pathogenic and
non-pathogenic bacteria. As such, their detection in a sample of drinking water
is an indication that the water is contaminated by sewage.
The presence of these bacteria is also very important because
the source of the bacteria is well known compared to the source of total
coliform bacteria (TC). Here, therefore, it becomes easier to locate and fix
the source of the problem and treat the water more effectively in order to prevent possible diseases associated.
* Fecal coliform bacteria can also be found in
such animals as shellfish. Therefore, people can get sick either by drinking
water contaminated by the bacteria or from eating contaminated shellfish.
of the illnesses that can result range from mild stomach upsets to severe
salmonellosis (salmonella poisoning) caused by salmonella bacteria.
E. coli is a sub-group of fecal coliform
bacteria and is largely composed of E. coli (Escherichia coli).
Compared to others, E. coli are almost exclusively found in the intestines of warm-blooded animals where they are able to live and reproduce.
Although they are mostly harmless in the host's intestines, there are strains
of E. coli (e.g. E.coli 0157:H7) that can cause serious illnesses. Detection of
these organisms in water is indicative of fecal contamination (recent contamination
in most cases) as well as possible presence of other pathogenic organisms that
may include viruses. In such cases, water is contaminated by sewage or animal
* E. coli cannot live long outside the host, for
this reason, their presence in water is evidence that water was recently
* If contaminated animal meat (such as beef) is
consumed (not cooked properly) it can cause the consumer to become ill
Types of Coliform
Citrobacter - A genus in
Enterobacteriaceae family that includes such bacteria as C. amalonaticus and C. freundii
Enterobacter - A genus in class Gammaproteobacteria
that includes Enterobacter cloacae and Enterobacter aerogenes
Hafnia - Also belonging to the
Enterobacteriaceae family, Hafnia includes such bacteria as Hafnia alvei
Escherichia - Genus of Family Enterobacteriaceae
and includes bacteria like E. coli
Some Coliform Characteristics
Gram negative - Coliform bacteria have a thin peptidoglycan layer and thus are
unable to retain the primary stain (gram staining) when washed with alcohol.
Typically, they will stain red or pink during staining because they take up the
counter stain. See Gram Stain
Do not form spores - During extreme conditions, some bacteria form spores so that
they can survive and germinate during favorable conditions. However, coliforms
do not undergo this process
They are facultative anaerobes- They can survive with very little or no oxygen
by using anaerobic respiration.
Rod shaped - Also known as bacillus, they are shaped like a rod (elongated)
They ferment lactose - Fermentation of lactose by coliform results in the production
of acid and gas, which helps in their identification during coliform testing
The following are some of the testing methods
used to determine whether total coliform bacteria are present in a sample of
Filtration (membrane filter
Water sample (ground or waste
Membrane filter - The
filter (cellulose ester membrane) used for this technique has pores of 0.45
micrometers and measures about 47milimeters
Poor about 100 milliliters
of the water sample through the filter
Place the filter on the
plate agar (on MI agar)
Incubate for about 24 hours
at 35 degrees (temperature)
For this technique, the filter membrane is used
to filter and thus retain any coliform bacteria that may be present in the
After incubation, the bacteria (if present) will use the nutrients in
the agar plate to grow.
If a blue color is observed, this indicates that the
beta-glucuronidase enzyme of E. coli was involved in breaking down
Indoxyl-beta-D-glucuronide (IBDG) in MI agar and thus indicates the presence of
A fluorescence appearance, however, indicates that
beta-galactosidase was involved in breaking down
4-methylumbel-liferyl-β-D-galactopyranoside (MUGal) which is also present in the
* For this technique, absorbent pads with lauryl
tryptose broth can also be used. These are transferred to either M-endo media
or agar to grow the bacteria.
* Once bacteria are cultures, the colonies are
then counted under the microscope.
Multiple Tube Fermentation Technique
(To determine the presence of rod shaped, facultative
anaerobic, gram-negative coliform group of bacteria that do not form spores)
Lauryl tryptose broth
For this technique, the procedure involves three
main phases that include:
Phase 1: Presumptive stage
Add lauryl trypsone broth
into several fermentation tubes
quantities of the sample (water sample) in to the tubes
Incubate the tubes for
between 24 and 48 hours at about 35 degrees Celsius (check every 24 hours for
* Gas formation (bubbles) in the tubes is marked
as a positive presumptive test
Phase 2: Confirmed state (using fermentation
tubes with brilliant green lactose bile broth)
For this step, only the
samples that marked positive in the first phase (positive presumptive test) are
To the fermentation tube
with the bile broth, inoculate a sample of the medium from the tubes that
tested positive in the first phase (immediately after gas formation)
Inoculate the tubes for about
48 hours at 35 degrees Celsius
* If gas is formed during this phase, it is an
indication of positive confirmed test
Phase 3: Completed test
For this phase, requirements include samples
with positive confirmed test, eosin methylene blue plate, incubator, lauryl
tryptose broth fermentation tube as well as nutrient agar slant.
Using a wire loop, scoop
and streak the methylene blue plates with the sample (this simply involved
marking out lines of the sample on the methylene blue plate, violet red agar
plate or MacConkey agar using a wire loop)
Incubate the plates for about
24 hours at 35 degrees Celsius
Obtain a colony of the
bacteria from the plate and transfer it to the fermentation tube (lauryl
tryptose broth) and nutrient agar slant
Incubate the two (agar
slants and fermentation tubes) for between 24 and 48 hours at 35 degrees Celsius to determine whether
any gas is produced
For the agar slant sample,
staining is required (gram-staining) in order to view the sample under the
microscope. As for the fermentation tube, check for presence of gas
* The presence of gas in fermentation tubes
indicates the presence of bacteria and this is marked satisfactory completed
test. If gram-negative, rod shaped (without spores)
bacteria are identified through microscopy, this is also indicative of the presence
of the bacteria (total coliform group)
For the agar plates, the media
used (culture media) inhibit the growth of gram positive bacteria and only
allow the test to determine their presence of gram-negative bacteria capable of
Depending on the media used, the color of the
agar plate will help indicate whether coliform are present in the sample:
MacConkey agar will turns
pink and cloudy indicating the presence of coliforms that ferment lactose
Eosin methylene blue agar
will show a metallic green sheen in the presence of coliforms
Violet red agar will turn
red or pink in color in the presence of coliform bacteria
MPN (Most Probable Number) Technique
Essentially, MPN is similar in principle to
multiple tube fermentation technique. However, rather than simply being used to
determine the presence of the bacteria (particularly fecal coliform) MPN is
used to estimate bacterial concentration in water in order to determine whether
the water is safe for use in homes.
As with multiple tube fermentation technique,
the technique involves three main steps (presumptive test, confirmatory test
and completed test). The sample is diluted in different tubes of different
sample concentration and inoculated in lactose broth. This technique makes it
possible to determine the amount of bacteria in different dilutions of the
sample after they are cultured.
The presence of the bacteria in the sample is
indicated by the production of either gas or acid (change in the color of the
media or presence of bubbles). For instance, all the tubes with the highest
concentration of the sample may test positive for the bacteria while a few of
the less concentrated tubes (less sample concentration) may prove positive
following the test.
The MPN index is used to show the number of
bacteria in the water and thus help determine whether the water is safe to
This involves the following steps:
For instance, if there were three sets of broth
tubes each with 5 tubes. Each set would be of different concentration. The first set (with 5 tubes) may be the original, undiluted sample,
the second set (with 5 tubes) may be 10 to the negative 1 dilution (half the
concentration of the original) while the third set (also with 5 tubes) may be
10 to the negative 2 dilutions (half the concentration of the second set).
Assuming that all of the tubes in set 1 are
positive of the bacteria, 3 in the second set are positive and only 1 in the
third set is positive, then these results can be compared to the MPN table to
determine the MPN index (estimated number of coliforms in 100mL of water) and
therefore determine whether the water is safe for use.
* MPN index below 2 is considered safe for
Microscopy can be used to view
E.coli coliform in wastewater, ground water or urine. Here, microscopy can be
used to view bacteria colonies or count individual bacterial cells.
Observing bacterial cells on a membrane
Membrane filter (with 0.45
A pair of forceps
A low power microscope
Sample (water, urine,
Prepared agar plate
* Before any step is taken, it is important to
ensure that all apparatus used are sterile. This helps prevent contamination
that can result in false results.
Place the funnel on the
collecting apparatus (the collecting apparatus may be connected to a vacuum
Place the filter below the
funnel so that it is between the funnel and the collecting apparatus
Pour the sample slowly into
the funnel and apply a vacuum in the collecting apparatus until the funnel is
If some sample is still in the
funnel, pour some sterile buffered dilution water (20-30ml) to rinse it
Apply the vacuum to empty
Using a pair of forceps, carefully remove the
membrane filter and place it on the agar plate in a Petri dish (MacConkey agar,
Eosin methylene blue agar and Violet red agar)
Cover the plate with a lid
and invert the plate
Incubate the plate for
about 2 hours at about 35 decrees Celsius
Using a low power
microscope, observe the coliform colonies on the plate (using 10 to 15x
Count the number of
individual colonies on the filter
* To determine the number of colonies in 100ml of
the sample, divide the number of colonies counted with the milliliters of the
sample used in the procedure and multiply the results with a 100. This will
give the percentage of the coliform in the water/urine or wastewater.
Fluorescent In Situ Hybridization Technique (FISH)
FISH (fluorescent in situ hybridization) refers
to a technique that uses fluorescent probes for the purposes of detecting and
comparing DNA sequences.
When it comes to detecting E.coli bacteria,
this technique has the advantage of saving time compared to the other
techniques commonly used for detecting the presence of coliform.
black polyester membrane
A pad (soaked in 80 percent
Hybridization buffer (50ul)
with respective probes
550 ul of washing
Filter the sample using the
0.40 um membrane filter
Transfer the filter the
ethanol soaked pad and allow to stand for about 3minutes at room temperature in
a Petri dish
Dry the filter membrane and
filter at room temperature for about 3 minutes
Place the membrane filter
on the hybridization buffer (50ul) in the hybridization chamber and incubate
for about 90 minutes at 46 degree
Place the membrane on the
washing buffer soaked buffer (550ul)
* for this technique, epifluorescence microscopy
is used to view the sample (with WIBA filter block)
Depending on the type of coliform present in the
sample, microscopy will show weak or high fluorescence intensity from the
fluorescent probes attached to the bacteria. This makes it possible to count
the number of individual bacteria in the specimen.
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