Apply US-EPA Wastewater ATP Guideline to Validate Modified Colitag for Enumeration of E.Coli and Fecal Coliform Bacteria

Collaborative Efforts to Improve Environmental Monitoring (Session 2)
Oral Presentation

Prepared by P. Biswas, L. Zhang
Neogen Corporation, 620 Lesher Place, Lansing, Michigan, 48912, United States

Contact Information: [email protected]; 517-372-9200


Colitag™, a ready-to-use medium, has previously been approved by the US-EPA as an Alternate Test Procedure (ATP) for simultaneous detection of total coliform bacteria and Escherichia coli in drinking water. In this study, the test protocol requirements set forth by the EPA Microbiological ATP Protocol was followed as guideline to validate the performance of Colitag medium coupled with multiwell MPNTray™ for enumeration of E. coli and fecal coliforms in wastewater samples. Detection is based on the presence of two enzymes, ß-glucuronidase and ß-galactosidase, which are characteristic of E. coli and coliform organisms, respectively.

A side-by-side test was conducted to assess comparability between the Colitag/MPNTray method, with an enumeration range of approximately 1-2000 MPN/100 mL, and the US-EPA approved reference method. Ten different wastewater matrices from ten different geographically diverse locations were collected for the study. Secondary sewage effluents from the sewage treatment plants were used as natural sources of fecal coliform and E. coli bacteria. Range finding analyses were performed to assess the concentration of target organisms in the matrix. The test samples for side-by-side comparison studies were generated by spiking of non-chlorinated final sewage effluent with appropriate level of secondary sewage effluent to obtain 20 of 100 mL wastewater samples per assay for each method. Positive or negative results were verified following Standard Method 9221 as descripted in section of the ATP guidelines. To evaluate the method comparability, recovery, precision, false positive and false negative rates were analyzed for each method using Standard Method 9221 as described in section of the ATP guidelines, and samples were further confirmed using the outlined procedure and protocols. The Colitag/MPNTray method demonstrated comparable quantitative results (MPN/100 mL) as well as comparable specificity and sensitivity in detecting E. coli and fecal coliform bacteria in wastewater, when compared to the reference method.