E.coli Easygel
Overview
For a refresher, this video is a great place to go.
E.coli, also known as Escherichia coli, is a bacterium that is found in the intestines of warm blooded animals. Scientists
use E. coli as an indicator of water that has been contaminated with fecal matter. The bacteria
associated with fecal matter (not just E. coli but many other types of bacteria and viruses) can make people sick if they come in
contact with contaminated water. Monitoring for E. coli helps protect water for recreation
and drinking water.
While wild animal waste going into lakes and streams cause naturally low levels of E. coli, the problem comes when too much untreated domestic animal or human waste enters water bodies. Problems can arise if waste from septic tanks, wastewater treatment plants, or animal production for food is not properly treated before it enters water ways.
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The Coliscan Easygel medium is a patented formulation for testing surface waters. It is a simple, accurate and quantitative way to identify and differentiate coliforms and E.coli from other bacteria in water. To identify the presence and numbers of coliforms and E.coli a sample is collected, then is added to the Easygel medium, mixed and poured into a petri-dish, and incubated at room temperature or at a higher controlled temperature (35oC is recommended). One of the main advantage of Coliscan Easygel method is that it uses proven and accepted technology to allow anyone to do effective coliform and E.coli testing without expensive equipment.
Note: As you collect your sample make observations that may give a clue to the source in case of contamination:
- Obvious pollution issues
- Visible discharge from pipes or outfall into the water
- Toilet paper and other materials commonly flushed
- Outfall pipes flowing during dry weather
- Patches of algal growth or vegetation
- Scum or films on or under outfall pipes
- Potential sources of bacteria such as pet, wildlife or livestock waste
- Any other unusual conditions
Below are general steps to the E.coli sampling, the tabs above have detailed explanations about how each step is performed:
1. In the field Sample Collection
2. Inside Plating
3. Incubation Period
4. Disposal
Identification guides: Easygel Guide (front), Easygel Guide (back), Supplemental Guide
Field Collection:
Steps |
Illustration |
| Pull off the top of the Whirl-Pak using the perforated line. |  |
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Use the white tabs to open the pouch; do not touch the top or inside of the bag.
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| Face upstream and collect your sample from a depth around 15 cm (6 in.). Be careful not to sample disturbed substrate or surface scum. |  |
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With bag open, in one quick motion, immerse the bag under water and fill making sure
to leave some head space.
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| Remove your bag from the water and quickly flip it over twice to secure it. Finish by twisting the yellow ties closed. |  |
| If you are monitoring more than one location record sample ID on bag. Water samples kept longer than 1 hour before plating need to be stored on ice. |
Inside Plating:
Steps |
Illustration |
| Make sure Coliscan Easygel medium in the bottle has thawed | |
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Strongly shake Whirl-Pak to thoroughly mix the sample. Carefully open bag using white tabs
so not to touch the lip or inside.
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Use a transfer pipe, and deposit 1 to 5 mL in the
Coliscan Easygel bottle, cap and gently swirl. Repeat for second duplicate sample. Be
sure to record how many mL you used.
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| Using a permanent marker record on the bottom of the petri dish the site id or name, date, time sample was plated, volume of sample, and sample #1 or 2. |  |
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Remove the lid, but be careful not to touch the inside of the petri dish or lid. Pour
Coliscan Easygel medium with sample water slowly into the bottom of the petri dish.
Make sure to coat entire bottom. Place the top on the petri dish and leave it on a
level surface out of direct sunlight for 45 minutes to solidify. Wash your hands.
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Incubation/Disposal
Incubation:
Steps |
Illustration |
| Store petri dishes in a warm draft free location. Use the thermometer to measure the
location. At room temperature, 20-24oC (68-75oF), colored colonies will take at least 48 hours to develop. Normally incubation
takes from 48-60 hours. Do not read samples after 72 hours, as bacteria can multiply,
resulting in inaccurate counts. |
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| Check every 10-12 hours to observe if colored colonies have started to form. Upon sighting the formation of colonies that are slightly colored, note the time and allow for another 24-30 hours for the maturation of these colonies. | |
| When you believe the colonies have matured (they will show strong coloration and the colonies will be visibly distinct) record the total number of hours on the data sheet in the Incubation time along with the normal temperature during incubation. |  |
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In a bright area, count ONLY colonies that have a dark blue or purple color. Dark blue and purple colonies indicate E.coli. Do not count pink, red, or teal, colonies as these indicate other Coliforms and different types of bacteria. Also ignore tiny, pin sized colonies. If you are unsure, look at examples provided. If still uncertain check with another UWW volunteer or take a photo and send to UWW staff. Volunteer Craig Page created a counting method to divide the dish into sections and count each one. This is a good strategy if there are a lot of colonies. Here are a couple of helpful guides: Easygel Guide (front), Easygel Guide (back), Supplemental Guide |
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| Record the number of dark blue or purple colonies on the data sheet for each sample. Enter the sample size (1 to 5) for each sample. Now you can calculate the E.coli per 100 mL (cfu). First divide 100 by the sample size. Then multiply this dilution factor by the colonies counted. |
Ex 1 (using 3 mL): 100mL / 3 mL Sample size = 33.33 33.3 X 9 colonies counted = 297 cfu / 100mL or Ex 2: (using 2 mL): 100mL / 2 mL Sample size = 50 50 x 9 colonies counted = 450 cfu / 100mL |
| Do this for both samples. Then average the two samples to the nearest whole number and record the average on the data sheet "Average E.coli cfu / 100 mL" |  |
Contact us (waterquality@usu.edu, 435-797-2580) if you numbers are above 400 cfu/100mL. Here is the health advisory flow chart for 2A and 2B waters.
Disposal:
Steps |
Illustration |
| Place a teaspoon of bleach onto the surface of the medium. Close the lid and let it sit for five minutes. | |
| Place both petri dishes in a water-proof (Ziploc bag) and throw in the trash. | |
| Wash your hands after handling samples, plates, and water. |