Microbiological Methods/Online Publications

Methods

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Bacteria

Method 1605: Aeromonas in Finished Water by Membrane Filtration Using Ampicillin-Dextrin Agar with Vancomycin (ADA-V) (October 2001) (PDF, 26 pp., 443 KB) - This method (EPA 821-R-01-034) describes a performance-based membrane filtration technique for the detection and enumeration of Aeromonas species. This method uses a selective medium that partially inhibits the growth of non-target bacterial species while allowing Aeromonas to grow and be detected by the production of acid from dextrin fermentation (causing a pH change producing yellow colonies). The method will be proposed for use in EPA's data gathering and monitoring programs under the Unregulated Contaminant Monitoring Rule.

Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium) (September 2002) (PDF, 18 pp., 486 KB) - This document (EPA 821-R-02-024) is identical to the February 2000 version of the MI Agar Method (Membrane Filter Method for the Simultaneous Detection of Total Coliforms and Escherichia coli in Drinking Water, EPA-600-R-00-13), with one exception, the addition of MI Broth. The MI Agar Method was approved for use in compliance monitoring of drinking water and source water in support of the National Primary and Secondary Drinking Water Regulations (Federal Register, Vol. 64, No. 230, 1 December 1999, p.67450-67467) becoming effective 3 January 2000. On 6 November 2001, MI Broth was approved as a minor modification. This method, now designated as Method 1604, has been approved for use in monitoring ambient water (Federal Register, Vol. 68, No. 139, 21 July 2003, p.43272-43283) becoming effective 4 August 2003. Four color photos of coliform and E. coli colonies on MI agar are also included in Method 1604. The procedure is in the format of the Environmental Monitoring Management Council (EMMC). The method may also be viewed as it appeared upon 1993 publication in the journal Applied and Environmental Microbiology (Vol 59, p.3534-3544), (EPA 600-J-99-225) but it does not include photos (2.2MB).

Method 1603: Escherichia coli (E.coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC) (September 2002) (PDF, 13 pp., 129 KB) - This method (EPA 821-R-02-023) combines information from a 1985 publication (Test methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure, EPA 600-4-85-076) and a subsequent March 2000 manual (Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli, EPA/821/R-97/004). Method 1603 is a revised membrane filter (MF) procedure, a single-step method that uses one medium, modified mTEC Agar, and does not require the transfer of the membrane filter to another medium or other substrate.

Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-ß-D-Glucoside Agar (mEI) (September 2002) (PDF, 14 pp., 125 KB) - This method (EPA 821-R-02-022) combines information previously published in the May 1997 version of Method 1600 (Membrane Filter Test Method for Enterococci in Water, EPA-821-R-97-004a) and a subsequent March 2000 manual (Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli, EPA/821/R-97/004). Method 1600 is a revision of EPA's previous enterococci method, used since 1985 in ambient water quality monitoring. It reduces analysis time to 24 hours and improves analytical quality. The method has been validated in single- and multi-laboratory studies and has undergone peer review.

Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA) (September 2002) (PDF, 16 pp., 806 KB) - This method (EPA 821-R-02-021) combines information from a 1985 publication (Test methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure, EPA 600-4-85-076) and a subsequent March 2000 manual (Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli, EPA/821/R-97/004). Method 1106.1 is a membrane filter (MF) procedure for the detection and enumeration of enterococci in water. The method incorporates the sequential use of a selective medium, mE agar, and a differential medium, EIA agar.

Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC) (September 2002) (PDF, 17 pp., 471 KB) - This method (EPA 821-R-02-020) combines information from a 1985 publication (Test methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure, EPA 600-4-85-076) and a subsequent March 2000 manual (Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli, EPA/821/R-97/004). Method 1106.1 is a membrane filter (MF) procedure for the detection and enumeration of E. coli in water. The method incorporates the use of a selective and differential medium, mTEC agar followed by incubation with urea substrate medium.

Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli (March 2000) (PDF, 49 pp., 350 KB) - This manual (EPA 821-R-97-004) describes four test methods for measuring bacteriological densities in ambient waters: the original and a revised method for detecting enterococci, and the original and a revised method for detecting E. coli. All four methods use a membrane filter procedure. The manual complements a training video on this topic but may be used independently. (To obtain the video, see pg 49 of the manual.)

Protozoans

Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA - December 2005 Update (PDF, 76 pp., 1.2 MB) - This method (EPA 815-R-05-002) is a performance-based method applicable to the determination of Cryptosporidium and Giardia in aqueous matrices. Method 1623 requires filtration, immunomagnetic separation (IMS) of the oocysts and cysts from the material captured, and immunofluorescent assay (FA) for determination of the oocyst and cyst concentrations. A Training module is also available.

Method 1622: Cryptosporidium in Water by Filtration/IMS/FA - December 2005 Update (PDF, 75 pp., 1.3 MB) - This method (EPA 815-R-05-001) is a performance-based method applicable to the determination of Cryptosporidium in aqueous matrices. Method 1622 requires filtration, immunomagnetic separation (IMS) of the oocysts from the material captured, and immunofluorescent assay (FA) for determination of the oocyst concentration. There are also forms associated with Method 1622. A Training module is also available.

Method 1622 and 1623 Forms - These forms are in PDF format and include text-only PDF versions for vision-assisted software: Bench Sheet (PDF, 1 pp., 9 KB) - text (PDF, 1 pp., 39 KB), Cryptosporidium report form (PDF, 1 pp., 55 KB) - text (PDF, 1 pp., 34 KB), Giardia report form (PDF, 1 pp., 15 KB) - text (PDF, 1 pp., 34 KB), Hemacytometer Data Sheet (PDF, 1 pp., 5 KB) - text (PDF, 1 pp., 30 KB), and Spike Enumeration Form (PDF, 1 pp., 57 KB) - text (PDF, 1 pp., 32 KB).

Criteria for Evaluation of Proposed Protozoan Detection Methods (PDF, 14 pp., 79 KB) - This document (EPA 815-K-99-02) provides a framework for evaluating methods for detecting Giardia, Cryptosporidium, and other protozoa.

Viruses and Coliphage

USEPA Manual of Methods for Virology - Twelve chapters. The individual chapters were prepared as ASCII text files to be viewed/printed with a monospaced font. It is planned to make them also available in PDF format in the future. The manual (EPA 600-4-84-013) describes in detail procedures for detecting viruses in environmental samples.

Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment Procedure (April 2001) (PDF, 40 pp., 463 KB) - This document (EPA 821-R-01-030) describes an enrichment procedure for detecting male-specific and somatic coliphage in groundwater and other waters.

Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure (April 2001) (PDF, 38 pp., 376 KB)- This document (EPA 821-R-01-029) describes an single agar layer procedure for detecting and quantitating male-specific and somatic coliphage in groundwater and other waters.

Recreational Water Publications

The EMPACT Beaches Project: Results From a Study on Microbiological Monitoring in Recreational Waters (PDF, 73 pp., 2.0 MB) - This report (EPA 600-R-04-023) describes the EMPACT (Environmental Monitoring for Public Access and Community Tracking) Beaches Project. The project was conducted to determine which physical, environmental and biological factors significantly influence the monitoring results of recreational waters. Project results will help beach managers and public health officials develop meaningful protocols for maintaining beach water safety.

Data Quality Objectives and Statistical Design Support for Development of a Monitoring Protocol for Recreational Waters (PDF, 31 pp., 100 KB) - This final report resulted from an expert workshop held in Cincinnati, Ohio to design a research plan that would be used to develop sampling guidance for monitoring water quality indicators in recreational waters.

Health-Based Monitoring of Recreational Waters: The Feasibility of a New Approach (The 'Annapolis Protocol') - 1999 (PDF, 50 pp., 1.3 MB) - This document presents the outcome of a meeting of experts assembled by the EPA and WHO in November 1998 in Annapolis, MD. The proceedings of the meeting describe a new approach to maintaining the quality of recreational water that better reflects health risk and provides enhanced scope for effective management intervention.

Health Effects Criteria for Marine Recreational Waters - This 1983 report (EPA 600-1-80-031) presents health effects water quality criteria for marine recreational waters. It can be used to develop guidelines based upon acceptable rather than detectable risk and is consistent with risk analysis. The document has been split into sections for easier downloading. Part 1 (PDF, 50 pp., 1.2 MB), Part 2 (PDF, 18 pp., 1.2 MB), Appendix Part 1 (PDF, 6 pp., 782 KB), App Part 2 (PDF, 10 pp., 1.1 MB), App Part 3 (PDF, 10 pp., 1.1 MB), App Part 4 (PDF, 10 pp., 1.1 MB), App Part 5 (PDF, 8 pp., 789 KB).

Health Effects Criteria for Fresh Recreational Waters (PDF, 33 pp., 1.3 MB) - This 1984 report (EPA 600-1-84-004) provides an assessment of the relationship between microbiological indicators of water quality and swimming-associated illness. The report resulted from a series of EPA-supported epidemiological-microbiological research projects designed to develop risk-based recreational water criteria for fresh waters.

ICR-related materials

Note: Sample collection for the Information Collection Rule ended in December, 1998. The following ICR publications are maintained online as informational resources.

ICR - Microbial Laboratory Manual (PDF, 233 pp., 1.2 MB) - This manual (EPA 600-R-95-178) is the "ICR Microbial Laboratory Manual" referenced in the Federal Register. It describes in detail the microbiolgical techniques used with the ICR.

Information Collection Requirements Rule - Protozoan Method for Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a Fluorescent Antibody Procedure (PDF, 59 pp., 2.8 MB) - This publication (EPA 814-B-95-003) is an illustrated guide for lab personnel analyzing water samples for Giardia and Cryptosporidium under the ICR. The guide complements a training video on this topic.

Information Collection Requirements Rule - Protozoa and Enteric Virus Sample Collection Procedures (PDF, 63 pp., 2 MB) - This publication (EPA 814-B-95-001) is an illustrated guide for the collection of water samples under the ICR. The guide complements a training video on this topic. Note: this June 1995 publication contains some material that has been changed with the May 1996 promulgation of the ICR.

ICR - MPNV Software Program (168 KB) - This program was used to perform the virus analyses for the ICR - calculating MPN (Most Probable Number) and confidence limits values (see the Virus Quantitation section, page VIII-27 of the ICR Microbial Laboratory Manual). This program is a DOS program. Save the file to a directory of your choice and (in DOS) type "MPNV" followed by " Enter." The file can also be run as a DOS application under Microsoft Windows® . See Example 1(page VIII-48 to VIII-55) and Example II (page VIII-57 to VIII-62) for examples of how to use the program.

Other Publications

Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples (PDF, 64 pp., 360 KB) - This publication (EPA 815-B-04-001) is intended to serve as a general guide for the development of laboratory and method-specific QA/QC procedures for PCR analysis of environmental analysis.

Preventing Waterborne Disease: A Focus on EPA's Research (PDF, 20 pp., 695 KB) - This publication (EPA 640-K-93-001) is heavily illustrated and describes microbiology-oriented research within the Office of Research and Development.

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Microbiological Methods/Online Publications

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