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

Collaborative Efforts to Improve Environmental Monitoring
Oral Presentation

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

Contact Information:; 517-372-9200


Modified Colitag™, a ready-to-use test, 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 for Drinking Water, Ambient Water, Wastewater, and Sewage Sludge Monitoring Methods was followed as guideline to validate the performance of Colitag medium using multi-well 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 between the Modified Colitag/MPNTray method, with an enumeration range of approximately 1-2000 MPN/100 mL, compared to the reference method (Standard method 9223, Colilert-18/Quanti tray 2000) was conducted. Non-chlorinated sewage effluents from several different sewage treatment plants were used as natural sources of coliform and E. coli bacteria. The test samples for evaluating this new method were generated using appropriate level of secondary sewage spikes diluted with final sewage effluent to obtain 100 mL wastewater sample per test. Recommended 20 sets of replicates were tested to generate data for the outlined statistical analysis. False positive and false negative rates were determined using Standard Method 9221 as descripted in section of the ATP guidelines, and samples were further confirmed using the outlined procedure and protocols. The Modified 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 reference method.