Jump to main content or area navigation.

Contact Us

Water: Bioindicators

Biological Pollutants in Ambient Water

new icon Revisions to Methods 1609 and 1611

On this page:


Microbiological Measurements

You will need Adobe Reader to view some of the files on this page. See EPA's PDF page to learn more.

In September 2008, we completed two reports:

Guidelines Establishing Test Procedures for the Analysis of Pollutants

Analytical Methods for Biological Pollutants in Wastewater and Sewage Sludge

September 29, 2007

We issued new methods that use culture-based approaches to detect enterococci and Escherichia coli (E. coli) in wastewater. Additional tests will also identify Salmonella and fecal coliform bacteria in sewage sludge.

CWA Methods of Interest Approved for use at 40 CFR Part 136
Number Method Title
1103.1 Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC) (PDF) (45 pp, 824K)
1106.1 Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA) (PDF) (41 pp, 755K)
1600 Membrane Filter Test Method for Enterococci in Water (PDF) (42 pp, 573K)
1603 Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC) (PDF) (42 pp, 504K)
1680 Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium (PDF) (50 pp, 172K)
1681 Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation using A-1 medium (PDF) (51 pp, 242K)
1682 Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium (PDF) (48 pp, 284K)

Top of page


Other Wastewater and Biosolids Methods

Note: Many of these methods are drafts.

Other CWA Methods of Interest not Currently Approved for use at 40 CFR Part 136
Number Method Title
200.7 Trace Elements in Water, Solids, and Biosolids by Inductively Coupled Plasma-Atomic Emission Spectrometry Revision 5.0 (PDF) (68 pp, 156K)  (EPA 821-R-01-010)
200.9 Trace Elements in Water, Solids, and Biosolids by Stabilized Temperature Graphite Furnace Atomic Absorption Spectrometry Revision 3.0 (PDF) (51 pp, 117K)  (EPA 821-R-01-011)
1656A Organo-Halide Pesticides in Wastewater, Soil, Sludge, Sediment and Tissue by GC/HSD Revision A (PDF) (58 pp, 150K)  (EPA 821-R-00-017)
1657A Organo-Halide Pesticides in Wastewater, Soil, Sludge, Sediment and Tissue by GC/FPD Revision A (PDF) (52 pp, 141K)  (EPA 821-R-00-018)
1683, Draft Specific Oxygen Uptake Rate in Biosolids Draft, January 2001 (PDF) (23 pp, 62K)  (EPA 821-R-01-014)
1684, Draft Total, Fixed, and Volatile Solids in Water, Solids, and Biosolids Draft, January 2001 (PDF) (16 pp, 41K) (EPA 821-R-01-015)
1685, Draft Nitrate/Nitrite-N in Water and Biosolids by Automated Photometry Draft, January 2001 (PDF) (28 pp, 67K)  (EPA 821-R-01-016)
1686, Draft Nitrate/Nitrite-N in Water and Biosolids by Manual Colorimetry Draft, January 2001 (PDF) (26 pp, 62K)  (EPA 821-R-01-005)
1687, Draft TotalKjeldahl Nitrogen in Water and Biosolids by Automated Colorimetry with Preliminary Distillation/Digestion Draft, January 2001 (PDF) (29 pp, 68K)  (EPA 821-R-01-004)
1688, Draft TotalKjeldahl Nitrogen in Water and Biosolids by Automated Colorimetry with Preliminary Semi-automatic Digestion Draft, January 2001 (PDF) (28 pp, 67K)  (EPA 821-R-01-007)
1689, Draft Ammonia-N in Water and Biosolids by Ion-Selective Electrode Potentiometry with Preliminary Distillation Draft, January 2001 (PDF) (25 pp, 67K) (EPA 821-R-01-012)
1690, Draft Ammonia-N in Water and Biosolids by Automated Colorimetry with Preliminary Distillation Draft, January 2001 (PDF) (27 pp, 65K) (EPA 821-R-01-009)
1693 Cryptosporidium and Giardia in Disinfected Wastewater by Concentration/IMS/IFA (PDF) (75 pp, 1.2MB) (EPA 821-R-14-013, September 2014)

Top of page


Rapid Microbiological Methods for Ambient Waters

new icon We are making available method revisions for EPA Methods 1609 and 1611 that use a genetic quantitative polymerase chain reaction (qPCR) approach to detect enterococci in ambient waters. The revised methods (1609.1 and 1611.1) include additional guidance and slight modifications.  The presence of a method here does not imply a regulatory requirement for its use.

These methods are not currently approved for use under 40 CFR Part 136
Number Method Title
1609.1 Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay (PDF) (71 pp, 655K) (EPA-820-R-15-099; April 2015)
Calculation Spreadsheet (Excel, 129K)
Changes to Method 1609: 
  • Section 9.11: Additional guidance is provided on the interpretation of ongoing calibrator sample CT values that coincides with modifications in the Method 1609.1/1611.1 calculation spreadsheet.
  • Section 9.13: More information is provided on the interpretation of Internal Amplification Control (IAC) assay results.
  • Section 11.1.8: A more standardized protocol is provided for preparing aliquots of cultured E. faecalis cells for use in subsequent preparation of calibrator and matrix spike samples.
  • Section 11.2: Slight modifications have been made in the protocol for preparing E. faecalis genomic DNA standards based on the modifications in Section 11.1.8.
  • Section 11.3: Modifications have been made in the protocol for preparing calibrator and matrix spike samples from aliquots of cultured E. faecalis cells to facilitate more standardized sample preparations. The new dilution protocol is based on the modifications in Section 11.1.8.
Changes to the calculation spreadsheet (note that the same spreadsheet can now be used for both Methods 1609.1 and 1611.1): 
  • The revised spreadsheet is now completely unlocked, which allows you to edit it.  (Note: use extreme caution in changing any of the existing calculation formulas).  In addition, the sequences column (column C) in the standards worksheet is populated with the titers of the current lot number of DNA standards that are available from EPA (see Appendix E in Methods 1609, 1609.1 & 1611.1).
  • Additional automatic calculations have been introduced to the spreadsheets for both methods to evaluate the quality of ongoing calibrator sample CT values and additional guidance has been provided for interpreting the variability (standard deviations) of initial calibrator sample CT values.
1611.1 Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (PDF) (72 pp, 668K) (EPA-820-R-15-008; April 2015)
Calculation Spreadsheet (Excel, 129K)
Changes to Method 1609: 
  • General: The format and content of this method has been modified to mirror that of Method 1609.1. The only substantial differences between these two methods are now in the Taq polymerase reagents (Sections 7.15), the recommended dilutions of water sample and corresponding calibrator sample DNA extracts for analysis (Sections 11.9.4 and Table 10) and in the interpretation of Internal Amplification Control (IAC) assay results (Sections 9.13).
  • Appendix E: This appendix has been added on how to request DNA standards at no charge from the EPA for a limited time.  These DNA standards can be used for all four methods (1609, 1609.1, 1611 and 1611.1).
  • For details regarding the calculation spreadsheet, please see Method 1609.1 above.
1609 Enterococci in Water by Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay (PDF) (72 pp, 988K) (EPA-820-R-13-005; March 2013)
Calculation Spreadsheet (Excel, 128K)
1611  Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (PDF) (56 pp, 413K) (EPA-821-R-12-008, October 2012)
Calculation Spreadsheet (Excel, 89K)
  Method B Bacteroidales in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (PDF) (111 pp, 8.6MB)

Videos About qPCR

This page provides links to non-EPA websites. You will leave the EPA.gov domain and enter another page with more information. Also, be aware that the privacy protection provided on the EPA.gov domain (see Privacy and Security Notice) may not be available at the external link. exit EPA
  • qPCR training videos developed by Michigan State University. Enterococci Testing Video Series: collecting samples, rules of sampling, lab supplies, lab safety, blank membrane filtration, water sample filtration, DNA extraction, preparing assay mix, and preparing tray
  • Southern California Coastal Water Research Project. Several videos, including qPCR training and microbial source tracking methods.
  • “qPCR in your lab” video from Water Environment Research Foundation. Introduces a rapid method for monitoring pathogen indicators.

Top of page


Jump to main content.