E.coli Easygel


E.coli, also known as Escherichia coli, is a bacterium that is found in the intestines of warm blooded animals. E.coli is a good indicator of health risks in fresh water and is currently recommended for testing of E.coli instead of fecal coliform bacteria.

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.

Below are detailed instructions to the E.coli Sampling:

  1. In the field Sample Collection
  2. Inside plating
  3. Incubation Period
  4. Disposal

In the field Sample Collection:

 

Steps

Illustration

Pull of the top of the Whirl-Pak using the perforated line. Do not touch inside the bag.
Gently pull the mouth of the bag open with the white tabs, making sure not to touch the top or inside the bag.
Face upstream and collect your sample from a depth around 15 cm (6 in.). Be careful not to sample disturbed substrate or surface scum.
Immerse the bag under water and fill making sure to leave some head space. 
Remove from water and quickly flip over twice to secure 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
Strongly shake Whirl-Pak to thoroughly mix the sample. Carefully open bag so not to touch the lip or inside. 
Use one transfer pipe for each sample, and deposit 1 to 5 mL in the Coliscan Easygel bottle, cap and gently swirl. Repeat for second sample.
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. 
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.

 

 

Incubation Period:
 

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 multiple, resulting in inaccurate counts.
Check every 10-12 hours to observe if colored colonies have started to form. Upon sighting the formation of slight colored colonies, note the time and allow for another 24-30 hours for the maturation of these colonies.
When you believe the colonies have matured (show strong coloration and distinct colonies) record the total number of hours on the data sheet in the Incubation time along with the normal temperature during incubation.
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. 
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: 100ml / 3 mL Sample size = 33.33

33.3 X 9 colonies counted = 297 cfu / 100mL

Do this for both samples. Then average the two sample to the nearest whole number and record on the data sheet Average E.coli sfu / 100 mL

 

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.