SOP Easy Gel
Preparing Samples
Start off by either collecting your water sample in a sterile container and transporting it back to the test
site, or take by taking the recommended 1–5 ml water sample directly from the source and placing it
directly into the bottle of Coliscan Easygel. Please Note: 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. Once inside the bottle, swirl
the bottle thoroughly for five seconds.
Preparando la Mezcla
Empiece con colectar su muestra de agua en un contenedor estéril y transportarlo de regreso al sitio de
ensayos, o introduzca de la fuente la muestra recomendada de 1-5 ml de agua directamente a la botella
de Coliscan Easy-Gel. Tenga en cuenta: muestras de agua que han sido guardadas por más de una (1)
hora antes de vertido, o cualquier botella de Coliscan Easygel que ha tenido una muestra por más de
diez (10 minutos), incluyendo el transporte, debe ser preservado en hielo o en el refrigerador hasta que
sea vertido. Una vez los líquidos han sido introducidos, agite la botella por cinco segundos.
Preparing Petri Dish
Once the petri dishes have been properly labeled, 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).The
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.
Preparando la placa de petri
Antes que deposites todo el liquido de la botella dentro de la placa, asegúrese que cada una este
marcada para distinguir entre ellas. Una vez las placas estén distinguidas y la botella sea mezclada, vacía
la botellita entera adentro de la placa. Asegúrese que no haya espacios vacíos, que toda la placa este
cubierta por el líquido de color café. Tenga cuidado de no derramar el líquido, y también no destape
completamente la cubierta de la placa. Trate de abrir suficiente para que el líquido pueda entrar. Es muy
importante que intento no hablar (las saliva es bastante volátil) o que el área dentro de la placa no se
contamine. Ya que el líquido este cubriendo la placa por dentro, deje que se quede en un lugar caliente,
30-37 C, para que el proceso de solidificación comience.
Incubation
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. The suggested temperature range is between 30-37 ̊ C
(85-99 ̊ F) .The coliform/E. coli organisms will grow faster at this temperature range than at room
temperature, so that results can be counted at 24-48 hours incubation time instead of about 24 hours
later if incubated at room temperature, 22-27 ̊ C (72-80 ̊ F).
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 2430 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 30-37 ̊ C, 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.
Incubation
E-Coli es una bacteria que tenemos de forma natural en nuestro organismo y es un muy buen indicador
para bacterias dañinas, por esta razón es muy importante tratar de encontrar E-Coli; donde se sabe que
existe también se pueden encontrar bacterias dañinas. E-Coli prefiere crecer en temperaturas más altas
que lo que normalmente se acostumbra para un cuarto, normalmente 22-270C.
Crecerán normalmente, pero crecerían más rápido si están entre 30-370C. En los laboratorios se usan
incubadoras, pero no es necesario para el campo. Hace diferencia tener los resultados en 24 horas y no
en 48.
Results
General coliforms will be a pink-magenta color. E. coli will grow as dark blue to purple colonies in the
medium; other bacterial types will generally grow as non–colored colonies. It is simple to count the
blue/purple colonies (E. coli) which indicate the number of E. coli per sample. The pink colonies indicate
the number of general coliforms per sample. The combined general coliform and E. coli number equals
the total coliform number. Any non-colored colonies which grow in the medium are not coliforms, but
may be members of the family Enterobacteriaceae. Since the Coliscan contains inhibitors, most other
bacterial types will not grow. Colonies which have the blue-green color 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 should also be ignored
Colonies 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.
In order to acquire a more quantitative result for the number of coliforms in your results:
a. Count 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 of
water.
b. Count 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.
Resultados
Después de 48 horas, se pueden distinguir circulitos de colores en las placas, estos círculos son colonias
de bacteria. Bacteria dañina apareceré con color rojo-rosado. E-Coli aparecerá como puntitos de color
azul-morado. Circulitos de color claro indican la presencia de más bacterias dañinas. Colonias de color
azul-verde no hacen parte de la familia de E-Coli, son otras bacterias dañinas. Lo más importante es
contar las colonias que tengan E-Coli; entre más E-Coli se encuentren, más contaminado esta tu agua.
Clean up and Possible Storage
Coliscan has a shelf life of 1 year and should be kept frozen until used. You may refrigerate for up to 2
weeks, but freezing is best in order to maintain color intensity throughout the 1 year period.
Do one of the following prior to disposal in normal trash:
a. Place dishes and Coliscan bottles in a pressure cooker and cook at 15 lbs. for 15
minutes. This is the best method.
b. Place dishes and Coliscan bottles in an ovenproof bag, seal it, and heat in an oven at
300° F for 45 minutes.
c. Places dishes and Coliscan bottles in a large pan, cover with water and boil for 45
minutes.
d. Place 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.
Guardando y Limpieza
La placa de Coliscan Easy-Gel puede mantener las colonias de bacteria y los colores, vivos hasta por un
año. Si desea mantener los resultados para más tiempo, se pueden congelar las placas hasta por dos
años. Congelando será la mejor manera para mantener la intensidad del color.
Para deshacerse de la placa hay varias opciones; recuerde que los contenidos dentro de la placa son
peligrosos. Primero, si tiene un aparato que deje cocinar en alta presión, introduzca las botellitas y las
placas dentro de la olla y cocine a cerca de 8 kilogramos de presión. La segunda opción es meter las
botellas y las placas dentro de una bolsa a prueba de hornos; debe sellarse y hornearse a 1490 por 45
minutos. La tercera opción, que es la más simple, es meter 5 ml de Blanqueador Clorox dentro de cada
placa, fijándose que cubre la placa entera; y deje la placa cerrada con el blanqueador por cinco minutos.
Después de los 5 minutos, introduzca todo entre una bolsa impermeable, que no pueda entrar ni salir
agua, para después tirar en la basura.
Sources
http://www.coastal.edu/wwa/vm/resource_files/Coliscan_Easygel_Instructions.htm
http://www.micrologylabs.com/Home/Our_Methods/Coliscan_Media/Coliscan_Easygel
Extra Info
Other coliform genera include Citrobacter, Enterobacter and Klebsiella. The USEPA acknowledges that E.
coli is the best indicator of health risk in fresh water and is currently recommending testing for E. coli
instead of fecal coliforms. The term "fecal coliform" indicates coliforms which will grow at a
temperature of 44.5 ̊ C. This is not an accurate designation as there are coliforms of non-fecal origin that
will grow at 44.5 ̊ C and there are strains of E. coli that will not grow at 44.5 ̊ C.
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.
*E. coli is the primary fecal coliform, however, Klebsiella is sometimes of fecal origin. Other general
coliform genera include Enterobacter and Citrobacter.
Non-fecal 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.
Coliform bacteria are members of the family Enterobacteriaceae and are defined as gram negative, nonspore-forming rods which ferment the sugar lactose with the evolution of gas and acids. Many coliforms
are normally found in soil and water and do not necessarily indicate the presence of fecal
contamination, but Escherichia coli (E. coli) is a primary bacterium in the human and animal intestinal
tract and its presence in food or water indicates fecal contamination. Therefore, E. coli is the coliform
that is used as an indicator for fecal contamination.
From Curt Maidman’s CE 350 Honors Report:
Methods. Concentration of all coliforms (not only fecal) in water can be tested using two methods.
These methods are the Coliscan Easygel Method and the Filtration Method. For the Coliscan Easygel
Method, you must first collect a sample of water from the source. You then add 1-5mL of that sample
to a bottle of Coliscan Easygel. That bottle must be mixed up, and then added into a sterile Petri dish.
This Petri dish is to be incubated at 32-37degreesC for 24 hours. After that time, observe the dish for
colonies of E. coli. Light pink colonies indicate non-fecal coliform, purple or blue colonies indicate fecal
coliform, and green colonies are disregarded as non-coliform bacteria(Hoosier, 2002).
For the filtration method, 1mL of the sample is to be added to 25mL of deionized water. A sterile
membrane filter must be placed on a filtration unit, and locked into place. The sample is then to be
filtered under a partial vacuum. Upon completion of the filtration, is to be placed onto a Petri dish, and
soaked with Coliscan MF medium. The sample is then incubated as described above. The colonies that
grow after a 24 hour period indicate the same type of bacteria is above also.
In order to form the colors that show up in the Petri dishes, certain enzymes that exist in most coliforms
are utilized. The main enzyme is Beta-D-glucuronidase (GUD). It is present in 94-96% of E. coli, but
some Salmonella as well. Because of this, the method is not %100 accurate. In order for the GUD to
form colors, it has to mixed with substrates. Some of the substrates used are p-nitrophenol-Beta-Dglucuronide (PNPG), 5-bromo-4-chloro-3-indolyl-Beta-D-glucuronide (X-GLUC) and 4-methylumbelliferylBeta-D-glucuronide (MUG). For example, when MUG is hydrolyzed with by GUD, is forms blue
florescence and it is known that E. coli is present. Also, when X-GLUC is hydrolyzed with GUD, it forms a
purple color, showing the presence of coliforms (Manafi, 2000).
References
Hoosier River Watch, 2002. E. Coli. IDEM Integrated Water Quality Monitoring and
Assessment Reponiesrt, IN.
Manafi, M., 2000. New Developments in chromogenic and fluorogenic culture media.
Elsevier Science, Vienna, Austria.
Wikipedia, 2006. Escherichia coli.
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SOP Easy Gel Preparing Samples Start off by either collecting your

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