Aerial view of the Warren, Maine lagoon system. Photo courtesy of Woodard and Curran.

Lagoon Systems In Maine 

Systems In Maine

An Informational Resource for
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Mars Hill Wastewater Lagoon System - Mars Hill  Maine. Photo Courtesy of Wright-Pierce Engineers.
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Coliform and E. coli Testing (2nd of 3 articles)  

Tim Loftus

         The first article in this series emphasized that bacteriological testing is not the same as chemical or physical analyses. Specifically, the validity of fecal coliform or E. coli results for NPDES reporting purposes is reflected by your Quality Assurance program; not by running spikes, standards, or knowns.

This article will cover certain federally required techniques and procedures needed to produce reportable results.

            All analyses for NPDES reporting must follow federal laws as outlined in 40 CFR 136. Approved test methods, collection and preservation techniques are outlined there. It is the final word. Where there is some ambiguity between a test method and 40 CFR, 40 CFR must be followed. An example of this is for bacteriological testing.  Federal law allows Standard Methods for the Examination of Water and Wastewater 18th edition, method  #9222D as one of the methods approved for fecal coliform analysis. In the supporting documentation for this in Standard Methods it outlines the preservation and holding time of a sample as follows: “Hold temperature of all…samples below 10 C during a maximum transport time of 6 hours. Refrigerate these samples upon receipt in the laboratory and process within 2 hours.” This translates into an 8-hour holding time at a temperature of less than 10 C. But 40 CFR states that preservation is to be at 4 C and the maximum holding time is 6 hours. This is an example where you must follow 40 CFR rather than the information in an approved source.

            When sampling for fecal coliform or E. coli, use sterilized glass or plastic containers. Do not rinse container with sample before collecting. Also, leave about an inch of headspace in the container to aid in mixing prior to aliquot removal. If there is any residual chlorine, you must dechlorinate with sodium thiosulfate. Note that this is a different dechlorinating agent than that used for dechlorinating BOD samples. Chlorinated BOD samples require sodium sulfite. Do not interchange the two.

Before setting up the equipment for analysis, wipe down the work area using antibacterial cleaning solutions. Always use equipment that has been sterilized at least 15 minutes at 15 psi and 121 C. Membrane filters and plastic sealing bags, for example, do not stand up to this heat and pressure sterilization process well. It is best to purchase these pre-sterilized where an alternative sterilization process is employed. Also, don’t forget to use aseptic techniques when mixing or transferring samples, rinsing equipment, and preparing samples for incubation. Aseptic techniques are techniques that eliminate or avoid contamination from the target organisms, and include such procedures as flaming the forceps before membrane filter transfers.

Prior to filtering your compliance samples, run a 100 mL buffered dilution water blank through the equipment. At the end of the incubation period, a negative result demonstrates that your equipment was not contaminated. A positive results invalidates the test and a new sample should be analyzed.

Finally, set up several dilutions for each sample. Make sure that the dilutions have a wide enough range so that at least one of these produces 20 to 60 colonies/filter for fecal coliform (20 – 80 for E. coli).

Record all the results, including the blank, on your bench sheets.

This article is just an overview of some of the techniques and procedures needed for successfully testing fecal coliform and E. coli. For specifics on making buffered dilution water, culture media, and other test procedures, refer to the instructions in your test method.

A third and final article in this series will cover the requirements for reading the results and how to calculate geometric means for NPDES reporting purposes.

            The information in this article is based on general test methods that are used throughout the New England area for NPDES monitoring of the fecal coliform group and for E. coli. As usual, check your federal, state, and local regulations. You may have additional regulations or reporting requirements that you must meet.

            If you have any questions, suggestions, or comments, please contact NEWEA Lab Practices Committee Chair Phyllis Arnold Rand 207-782-0917  ( or Tim Loftus at (508) 949-3865 (






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