Parameter and units | Method 1 | EPA | Standard Methods33 | AOAC, ASTM, USGS | Other |
1. Coliform (fecal), number per 100 mL | Most Probable Number (MPN), 5 tube, 3 dilution, or | p. 1323 | 9221 E-2014, 9221 F.2-2014 32 | | |
| Membrane filter (MF),2 single step | p. 1243 | 9222 D-201526 | B-0050-854 | |
2. Coliform (total), number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1143 | 9221 B-2014 | | |
| MF,2 single step or two step | p. 1083 | 9222 B-201527 | B-0025-854 | |
| MF2 with enrichment | p. 1113 | 9222 (B + B.4e)-201527 | | |
3. E. coli, number per 100 mL | MPN5,7,13, multiple tube, or | | 9221 B.3-2014/9221 F-201410,12,32 | | |
| Multiple tube/multiple well, or | | 9223 B-201611 | 991.15 9 | Colilert®,11,15 Colilert-18®11,14,15 |
| MF2, 5, 6, 7, two step, or | 1103.118 | 9222 B-2015/9222 I-2015,17 9213 D-2007 | D5392-938 | |
| Single step | 160319 160420 | | | m-ColiBlue24®16, KwikCountTM EC28,29 |
4. Fecal streptococci, number per 100 mL | MPN, 5 tube, 3 dilution, or | p. 1393 | 9230 B-2013 | | |
| MF2, or | p. 1363 | 9230 C-201330 | B-0055-854 | |
5. Enterococci, number per 100 mL | MPN5,7, multiple tube/multiple well, or | | 9230 D-2013 | D6503-998 | Enterolert®11,21 |
| MF2, 5, 6, 7 two step, or | 1106.122 | 9230 C-201330 | D5259-928 | |
| Single step, or | 160023 | 9230 C-201330 | | |
6. Cryptosporidium | Filtration/IMS/FA | 162224, 162325, 1623.125,31 | | | |
7. Giardia | Filtration/IMS/FA | 162325, 1623.125,31 | | | |
1 The method must be specified when results are reported.
2 A 0.45-μm membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of extractables which could interfere with their growth.
3 Microbiological Methods for Monitoring the Environment, Water and Wastes. EPA/600/8-78/017. 1978. U.S. EPA.
4 U.S. Geological Survey Techniques of Water-Resource Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples. 1989. USGS.
5 Tests must be conducted to provide organism enumeration (density). Select the appropriate configuration of tubes/filtrations and dilutions/volumes to account for the quality, character, consistency, and anticipated organism density of the water sample.
6 When the MF method has not been used previously to test waters with high turbidity, large numbers of noncoliform bacteria, or samples that may contain organisms stressed by chlorine, a parallel test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of results.
7 To assess the comparability of results obtained with individual methods, it is suggested that side-by-side tests be conducted across seasons of the year with the water samples routinely tested in accordance with the most current Standard Methods for the Examination of Water and Wastewater or EPA alternate test procedure (ATP) guidelines.
8 Annual Book of ASTM Standards—Water and Environmental Technology. Section 11.02. 2000, 1999, 1996. ASTM International. 9 Official Methods of Analysis of AOAC International, 16th Edition, Volume I, Chapter 17. 1995. AOAC International. 10 The multiple-tube fermentation test is used in 9221B.3-2014. Lactose broth may be used in lieu of lauryl tryptose broth (LTB), if at least 25 parallel tests are conducted between this broth and LTB using the water samples normally tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total coliform using lactose broth is less than 10 percent. No requirement exists to run the completed phase on 10 percent of all total coliform-positive tubes on a seasonal basis.
11 These tests are collectively known as defined enzyme substrate tests.
12 After prior enrichment in a presumptive medium for total coliform using 9221B.3-2014, all presumptive tubes or bottles showing any amount of gas, growth or acidity within 48 h ± 3 h of incubation shall be submitted to 9221F-2014. Commercially available EC-MUG media or EC media supplemented in the laboratory with 50 μg/mL of MUG may be used.
13 Samples shall be enumerated by the multiple-tube or multiple-well procedure. Using multiple-tube procedures, employ an appropriate tube and dilution configuration of the sample as needed and report the Most Probable Number (MPN). Samples tested with Colilert® may be enumerated with the multiple-well procedures, Quanti-Tray® or Quanti-Tray®/2000, and the MPN calculated from the table provided by the manufacturer.
14 Colilert-18® is an optimized formulation of the Colilert® for the determination of total coliforms and E. coli that provides results within 18 h of incubation at 35 °C, rather than the 24 h required for the Colilert® test, and is recommended for marine water samples.
15 Descriptions of the Colilert®, Colilert-18®, Quanti-Tray®, and Quanti-Tray®/2000 may be obtained from IDEXX Laboratories Inc.
16 A description of the mColiBlue24® test may be obtained from Hach Company.
17 Subject coliform positive samples determined by 9222B-2015 or other membrane filter procedure to 9222I-2015 using NA-MUG media.
18 Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC), EPA-821-R-10-002. March 2010. U.S. EPA.
19 Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC), EPA-821-R-14-010. September 2014. U.S. EPA.
20 Method 1604: Total Coliforms and Escherichia coli (E. coli) in Water by Membrane Filtration by Using a Simultaneous Detection Technique (MI Medium), EPA 821-R-02-024. September 2002. U.S. EPA.
21 A description of the Enterolert® test may be obtained from IDEXX Laboratories Inc.
22 Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA), EPA-821-R-09-015. December 2009. U.S. EPA.
23 Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI), EPA-821-R-14-011. September 2014. U.S. EPA.
24 Method 1622 uses a filtration, concentration, immunomagnetic separation of oocysts from captured material, immunofluorescence assay to determine concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the detection of Cryptosporidium. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA, EPA-821-R-05-001. December 2005. U.S. EPA.
25 Methods 1623 and 1623.1 use a filtration, concentration, immunomagnetic separation of oocysts and cysts from captured material, immunofluorescence assay to determine concentrations, and confirmation through vital dye staining and differential interference contrast microscopy for the simultaneous detection of Cryptosporidium and Giardia oocysts and cysts. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. EPA-821-R-05-002. December 2005. US EPA. Method 1623.1: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. EPA 816-R-12-001. January 2012. U.S. EPA.
26 On a monthly basis, at least ten blue colonies from positive samples must be verified using Lauryl Tryptose Broth and EC broth, followed by count adjustment based on these results; and representative non-blue colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.
27 On a monthly basis, at least ten sheen colonies from positive samples must be verified using Lauryl Tryptose Broth and brilliant green lactose bile broth, followed by count adjustment based on these results; and representative non-sheen colonies should be verified using Lauryl Tryptose Broth. Where possible, verifications should be done from randomized sample sources.
28 A description of KwikCount™ EC may be obtained from Micrology Laboratories LLC.
29 Approved for the analyses of E. coli in freshwater only.
30 Verification of colonies by incubation of BHI agar at 10 ± 0.5 °C for 48 ± 3 h is optional. As per the Errata to the 23rd Edition of Standard Methods for the Examination of Water and Wastewater, “Growth on a BHI agar plate incubated at 10 ± 0.5 °C for 48 ± 3 h is further verification that the colony belongs to the genus Enterococcus.”
31 Method 1623.1 includes updated acceptance criteria for IPR, OPR, and MS/MSD and clarifications and revisions based on the use of Method 1623 for years and technical support questions.
32 9221 F.2-2014: This procedure allows for simultaneous detection of E. coli and thermotolerant coliforms by adding inverted vials to EC-MUG; the inverted vials collect gas produced by thermotolerant coliforms.
33 Standard Methods for the Examination of Water and Wastewater, Joint Editorial Board, American Public Health Association, American Water Works Association, and Water Pollution Control Federation, 23rd Edition (2017), 22nd Edition (2012), 21st Edition (2005), 20th Edition (1998), 19th Edition (1995), and 18th Edition (1992).
Section 16. Effective Date. This rule takes effect on the first day of the month following publication in the Wisconsin Administrative Register as provided in s. 227.22 (2) (intro.), Stats. Section 17. Board adoption. This rule was approved and adopted by the State of Wisconsin Natural Resources Board on ________________.
Dated at Madison, Wisconsin _____________________________.
STATE OF WISCONSIN
DEPARTMENT OF NATURAL RESOURCES
BY ______________________________________
Preston D. Cole, Secretary
(SEAL)
