Coliform bacteria testing: a how to

Coliform bacteria are associated with water that is unfit to drink as they come from fecal matter. Ideally, our drinking water would have no coliform bacteria in it, otherwise, water should be boiled to drink. Being able to perform your own coliform bacteria test is ideal if you are a well owner or have a water body on your property. It is fairly easy to do but does require some startup cost. There is a growing movement of DIY Biolabs that may have equipment available for use. The method that I will be outlining is the membrane filter method (EPA method 1604). In this method, water samples are filtered through a membrane that retains bacteria and then the samples are placed on growth medium and incubated. The bacteria grow dome-shaped colonies which are then counted after 24 hours. Please note that I am not an expert, and this article should not be used entirely for testing. Please read the EPA method and if you feel you don’t fully understand the method then have your drinking water tested by a lab.


Collect 100 mL of a sample in a sterile container and try to make sure that no large particles are in the sample as they will clog the filter. In a vacuum filter system place a  0.45-µm pore size cellulose ester membrane filter. Filter the sample and place the filter 5-mL plate of MI agar or on an absorbent pad saturated with 2-3 mL of MI broth. Be sure that the plate is sterile, and that the filter is moved using sterile forceps (these can be sterilized by dipping in ethanol and lighting on fire). The plate is incubated at 35°C for up to 24 hours. Blue colonies that form are E. coli, others are different types of coliform bacteria. Use a microscope with 10-15X to count the colonies. Calculate the number of colonies per 100 mL of both E. coli and total coliform bacteria.

EPA Method 1604

Here is some data that I collected from well water and a local creek as well as my analysis.

The Petri dishes were set up on April 18th, 2014 at 12:00 pm in the laboratory at Alfred University in Alfred, NY using membrane filtration methods using a medium that only displays total coliform counts. Two samples were analyzed. One came from Dr. Fred Beadury’s well in Alfred, NY. The other sample can from the Kanakadea Creek near the area where the bandstand was be taken down. Both samples were analyzed within 6 hours of collection and were stored in a freezer until being analyzed. Colonies were counted on April 19th, 2014 at 9:38 am. The number of colonies was calculated using 100 mL of sample and then counting the colonies under a magnification of 13 X.

Table 1. Results of coliform analysis on waters from Alfred New York in April 2014.

  Sample 1- Fred’s Well Water Sample 2- Kanakadea Creek Water
Total number of coliform colonies 0 colonies per 100 mL 168 colonies per mL


It is no surprise that the well water had no fecal coliform bacteria colonies as it is not allowed to have any and would need to be retested if any was found. If those following tests were positive then the water would need to be treated either by chemicals (chlorination), boiling, ultra-violet light, or exposure to ozone. In addition, the bacteria need nutrients to survive which there are very few in groundwater. The well meets the criteria for class AA water in regards to coliform bacteria. Class AA waters are used for drinking water, food processing and cooking, and fishing. They are suitable for both primary and secondary contact.

Since the creek water had less the 200 colonies it is likely that it is not pathogenic. The possible sources of contamination could be either natural (from the soil) or caused by warm-blooded animals. There is a large population of cats that frequent the dumpsters near a local restaurant, therefore, it is possible that they use the creek for drinking water and they could have defecated near it leading to these levels. In addition, it is also possible that the other wildlife of Alfred such as deer, raccoons, and skunks also use the creek and therefore also defecate near it. The creek meets the requirements for class A water in regards to coliform bacteria. Class A waters are used for drinking water, food processing and cooking, and fishing. They are suitable for both primary and secondary contact.

Shopping list: Total cost $897

50 pack MI Broth- $137

Bacteria Vacuum Filter System- $274

Membranes- $21

Handpump- $22

Incubator- $300

Microscope- $136

Petri Dishes- $7

I know these are huge start-up costs but I also found this kit for $25 dollars Testing kit

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