Detection of Waterborne Coliforms and E. coli with Coliscan Easygel
TheColiscan Easygel medium is a patented formulation
for water testing. It contains a sugar linked to a dye which, when
acted on by the enzyme ß-galactosidase (produced by coliforms
including E. coli), turns the colony a pink color. Similarly,
there is a second sugar linked to a different dye which produces a
blue-green color when acted on by the enzyme ß-glucuronidase.
Because E. coli produces both ß-galactosidase and
ß-glucuronidase, E. coli colonies grow with a purple
color (pink + blue). The combination of these two dyes makes possible
the unique ability to use one test to differentiate and quantify
coliforms and E. coli. (Because E. coli is a member of
the coliform group, add the number of purple colonies to the number of
pink colonies when counting total coliforms.)
Either collect your water sample in a sterile container
and transport the water back to the test site, or take a measured water
sample directly from the source and place directly into the bottle of Coliscan
Easygel. Water samples kept longer than one (1) hour prior to
plating, or any Coliscan Easygel bottle that has had a
sample placed into it for transport longer than ten (10) minutes,
should be kept on ice or in a refrigerator until plated.
the petri dishes with the appropriate sample information. A permanent
marker or wax pencil will work.
transfer water from the sample containers into the bottles of ColiscanEasygel (Consult the following table for rough
guidelines for inoculum amount). Swirl the bottles to distribute the
inoculum and then pour the medium/inoculum mixtures into the correctly
labeled petri dishes. Place the lids back on to the petri dishes.
Gently swirl the poured dish until the entire dish is covered with
liquid (but be careful not to splash over the side or on the lid).
Inoculation of Coliscan
river, lake, pond, stream, ditch
1.0 to 5.0 mL
well, municipal, bottled
dishes may be placed right-side-up directly into a level incubator or
warm level spot in the room while still liquid. Solidification will
occur in approximately 40 minutes.
at 35° C (95° F) for 24 hours, or at room temperature for 48
hours. (See comments on incubation)
all the purple colonies on the Coliscan dish (disregard any
light blue, blue-green or white colonies) , and report the results in
terms of E. coli per ml of water. NOTE: To report in terms of E.
coli per 100 ml of water, first find the number to multiply by. To
do this: first, divide 100 by the number of ml that you used for your
sample. Then, multiply the count in your plate by the result obtained
from #1. For example, a 3 ml sample, 100 / 3 = 33.3. So, 4 E. coli
colonies multiplied by 33.3 will equal 133.2 E. coli per 100 ml
all the pink and purple colonies on the Coliscan dish
(disregard any light blue, blue-green or white colonies) and report the
results in terms of coliforms per ml of water.
one of the following prior to disposal in normal trash:
dishes and Coliscan bottles in a pressure cooker and cook at 15
lbs. for 15 minutes. This is the best method.
dishes and Coliscan bottles in an ovenproof bag, seal it, and
heat in an oven at 300° F for 45 minutes.
dishes and Coliscan bottles in a large pan, cover with water
and boil for 45 minutes.
5 ml (about 1 teaspoon) of straight bleach onto the surface of the
medium of each plate. Allow to sit at least 5 minutes. Place in a
watertight bag and discard in trash.
on Incubation: Micrology Laboratories, LLC in-house studies indicate
that Coliscan can effectively differentiate general coliforms
from E. coli when incubated at either room temperatures or at
elevated temperatures (such as 90-98° F). However, some further
explanation may be helpful.
is no one standard to define room temperature. Most would consider
normal room temperature to vary from 68-74° F, but even within this
range the growth of bacteria will be varied. Members of the bacterial
family Enterobacteriaceae (which includes coliforms and E.
coli*) are generally hardy growers that prefer higher than room
temperatures, but which will grow at those temperatures. They tend to
grow at a faster rate than most other bacterial types when conditions
are favorable. It is therefore logical to try to place inoculated
dishes in a "warm" place in a room for incubation if a controlled
temperature incubator is not available. It is a very easy task to make
an adequate incubator from a box with a 40-60 watt bulb in it to
provide heat at an even rate. One can also use a heat tape such as it
is used to prevent the freezing of pipes in the winter as your heat
general instructions indicate that incubation times for coliforms
(including E. coli) are generally 24-48 hours at elevated
temperatures (90-98° F) and 48 or more hours at room temperatures.
At elevated temperatures, no counts should be made after 48 hours as
any coliforms present will be quite evident by that time and if new
colonies form after 48 hours as any coliforms present will be quite
evident by that time and if new colonies form after 48 hours they are
most likely not coliforms, but some other type of slow growing
organisms that should not be included in your data. At room
temperatures, the best procedure is to watch the plates by checking
them at 10-12 hour intervals until you observe some pink or purple
colonies starting to form and then allowing another 24-30 hours for the
maturation of those colonies. Since the coliforms (including E. coli)
are generally the faster growing organisms, these will be the first to
grow and be counted. Colonies that may show up at a later time are
likely to not be coliforms. As you can see, there are advantages to
incubating your dishes at elevated temperatures. First, you can count
the results earlier. At 95° F, it is often possible to do accurate
counts at 18-20 hours of incubation. There is also less probability of
variation from batch to batch when the incubation temperatures are kept
at one uniform level. And a higher incubation temperature will tend to
inhibit the growth of non-coliforms that may prefer lower temperatures.
coli is the primary fecal coliform, however, Klebsiella is
sometimes of fecal origin. Other general coliform genera include
Enterobacter and Citrobacter.
of Results This test method utilizes well established, widely
accepted criteria for the recognition of coliforms and E. coli
and proper application of the method will result in accurate results.
Therefore, if you suspect that your water is dangerously contaminated
based on the results you get using Coliscan Easygel, you should
contact your local health department and ask for their help in
performing an official assessment of water.
coliforms are widely distributed in nature, being found both as
naturally occurring soil organisms, and in the intestines of
warm-blooded animals and humans. Fecal coliforms are coliforms found
naturally only in the intestines of warm-blooded animals and humans.
Fecal coliform contamination is therefore the result of some form of
fecal contamination. Sources may be either animal or human.
Notes on Differentiating Coliforms and E. coli Generally, water containing E. coli (the
fecal contamination indicator organism) should not be used for drinking
water unless it is sanitized in some manner. Contact your local health
department for guidelines regarding E. coli and coliforms in
recreational waters. Inform them if you suspect that contamination may
be occurring from a specific source.
which have the blue-green color are not exhibited any
ß-galactosidase activity (which is evidenced by the pink color).
Because of this, they are not considered to be either coliforms or E.
coli and therefore should be ignored when counting your coliform or
E. coli colonies. Similarly, colonies which are
white are exhibiting neither color-causing enzyme, and should also be
on the surface of the plate are exposed to the medium on only the
underside of the colony. This causes these colonies to appear with much
less of the indicator color. E. coli colonies may only have a
slight purple tinge to them, and it may appear only in the center of
the colony with the remainder of the colony being white. Similarly,
coliforms on the surface may be light pink or white with a pink center.