Demonstration of EPA Method 1603 - Modified m-TEC for E. coli

EPA 1603: E. coli by Modified m-TEC

The purpose of this illustration is to demonstrate a great new laboratory method for the enumeration of E. coli bacteria. This method, EPA 1603, has been around for a few years, but was recently (March, 2007) proclaimed one of only three methods approved by the EPA for the enumeration of E. coli by a permitted (NPDES -National Primary Discharge and Elimination) wastewater treatment plant.

Using Modified m-TEC is an advantage over traditional m-TEC in that it is a single step procedure. Incubation for both is the same, 35 +/- 0.5 degrees C. for 2 hrs., then 44.5 degrees C. +/- 0.2 for 22 hrs. With regular m-TEC, you need to remove the filter from the plate at the end of the incubation period and place it in a petri dish bottom onto a pad soaked with urea substrate. Allow fifteen minutes for the reaction to occur, then count the yellow-brown-tan colonies that form. These are E. coli.

With Modified m-TEC, after incubation, you just count the bright red-magenta colonies that form. These are E. coli. The Modified m-TEC media is very good at what it was designed for because it is very selective for E. coli - encourages the growth of E. coli, and differential against competing bacteria - discourages the growth of other competing organisms.

Modified m-TEC USEPA 1603 Getting Great Results!

Modified m-TEC The Advantages

Modified m-TEC with E. coli Colonies

Modified m-TEC makes colonies  turn a nice Magenta color which confirms E. coli.
Modified m-TEC makes colonies turn a nice Magenta color which confirms E. coli. | Source

Membrane Filter

Membrane filter being placed onto magnetic filter funnel base for E. coli filtration and identification by Modified m-TEC methodology.
Membrane filter being placed onto magnetic filter funnel base for E. coli filtration and identification by Modified m-TEC methodology.

Wet Bath with Gable Cover Incubator at 44.5 C.

Wet bath with gable cover for E. coli incubation at 44.5 degrees C. for 22 hrs.  Also a great hot tub for elves!!
Wet bath with gable cover for E. coli incubation at 44.5 degrees C. for 22 hrs. Also a great hot tub for elves!!

Membrane Filtration

As you can see here, the E. coli colonies are bright red-magenta and are easy to see and count. When performing the membrane filtration procedure for E. coli for a wastewater treatment or pollution control facility, the sample should be run (filtered and placed in the incubator), within 6 hours from the time the sample has been taken. The first step in doing a membrane filtration, once the sample has been taken (100 mLs. or about 4 ozs.), is to autoclave the filter funnels to sterilize them. This is done at 15 psi. and 121 degrees C. for at least fifteen minutes per cycle in the autoclave.

Once sterilized (which is the absence of all living things), as opposed to sanitized (which is the significant reduction of germs), the filter funnels are placed on the vacuum manifold which holds the funnels securely over the membrane filter by a magnet in the base. The funnels are rinsed several times around the inside with sterile dilution water, then the sample is poured into the funnel and filtered through. If the sample is turbid (cloudy) and doesn't filter through within fifteen minutes or so, repeat using only 50 mLs. of sample and multiply your answer (in CFU/ 100 mLs.) by two. Rinse funnels again with sterile water to wash any extraneous bacteria onto the filter from the funnel wall.

After filterering, remove the funnel and then the membrane filter and place the filter onto the prepared Modified m-TEC media plate. Run positive and sterile controls, then place upside down in dry incubator at 35.0 +/- 0.5 degrees C. for two hours. Transfer to a wet bath incubator with gable cover at 45.0 +/- 0.2 degrees C. for 22 hours. Remove and count the bright red-magenta colonies. That's it! Reporting E. coli colonies is done by CFU/ 100 mLs., (colony forming units per 100 milliliters because each individual bacteria gives rise to a full colony (millions of bacteria) which can be easily seen and counted. The special chromogen (5-bromo-6-chloro-3-indolyl-B-D-glucuronide) in the media is metabolized by the E. coli and gives off a red chromogenic product resulting in the colonies being colored red-magenta.

Modified m-TEC represents a step in the right direction with respect to providing fast, accurate, and easy to read results for wastewater professionals, laboratories, recreational facilities, and myriad other groups that depend on safe water for drinking and recreational use. And with the bright red-magenta colonies - E. coli never looked so good! (sorry - I just had to say that!). I'm usually pretty busy at my lab - Aquacheck, but glad to answer any questions you have about Modified m-TEC or E. coli in general. Thanks for reading!

Scott Bradley

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Modified m-TEC - The Advantages 1 comment

Anonymous 5 years ago

very good website but should put more information on instructions and why its important

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