Ohio Water Microbiology Laboratory

U.S. Department of the Interior
U.S. Geological Survey

Fact Sheet 2005-3072

Overview

The U.S. Geological Survey (USGS) Ohio Water Microbiology Laboratory (OWML) addresses water-related public-health concerns for Ohio and the rest of the Nation. The OWML works with government agencies, academic institutions, and other partners to study the quality of national, state, and local water resources. The OWML is involved in investigations of processes that affect microorganisms in the environment and testing of new methods to improve detection and interpretation of microbiological presence in water.

The OWML is located in the USGS Columbus, Ohio, office and consists of a 1,000-ft² main laboratory area and a 300-ft² limited-use area. The main area is used for sample login and preparation, media and reagent preparation, membrane filtration, incubation, culture maintenance, and reagent preparation for molecular methods. The limited-use area is the only area in the building where products from molecular methods are manipulated. The limited-use area is kept separate from the main area to prevent cross-contamination of incoming samples. The OWML is equipped to conduct a wide variety of cultivation- and molecular-based microbiology methods, including membrane filtration, direct microscopy, polymerase chain reaction, and hybridization with gene probes.

Mission

The OWML provides microbiological data of public-health significance from surface waters, ground waters, and sediments for a variety of study objectives. The goals of the OWML are to

provide quality microbiological analytical services to USGS projects for the analysis of environmental samples for bacterial indicators, coliphage, enteric viruses, and protozoa.
work with other government agencies, academic institutions, and public utilities to develop and (or) test methods for detection or enumeration of microorganisms of public-health significance in the environment.
provide assistance to other USGS employees for project planning and training related to microorganisms of public-health significance.
continually develop projects and programs that enhance our understanding of the processes that affect microorganisms in the environment.

Photograph showing Ohio Water Microbiology Laboratory technician analyzing a water sample for coliphage using a single-agar layer technique.

OWML laboratory technician analyzing a water sample for coliphage using a single-agar layer technique

OWML Capabilities

The OWML provides water-quality data on three major groups of microorganisms of public-health significance in the United States–bacteria, viruses, and protozoa. Surface water, ground water, and sediments are analyzed for these microorganisms. Pathogenic (disease-causing) organisms are of primary concern in our Nation’s waters. Methods to detect these organisms are often costly and time consuming, thus, it is impractical to monitor directly for pathogens by traditional methods. Indicator organisms are used to assess the microbiological quality of water and provide information on the possible presence of pathogens. Indicator organisms are microorganisms that are associated with the intestinal tract of warmblooded animals and are consistently present in fecal waste. They occur in greater numbers than the associated pathogens of interest, and their presence in water indicates fecal contamination of the water. Descriptions of common bacterial and viral indicator organisms are given in the following pages of this fact sheet, as well as the methods used by the OWML to detect them. Also described are viral and protozoan pathogens and the detection methods used by the OWML.

Bacterial Indicators

Coliforms

Total coliforms are found in animal intestines, in soils, on vegetation, and in industrial wastes. They are used to assess drinking-water or ground-water quality.
Fecal coliforms are total coliforms that are able to grow at elevated temperatures and are often, but not always, of fecal origin.
Escherichia coli (E. coli), a member of the fecal coliform group, is a natural inhabitant of the gastrointestinal tract of warmblooded animals and is direct evidence of fecal contamination.

Streptococci

Fecal streptococci are found in feces; however, some species live on plants and in soil.
Enterococci are a subset of fecal streptococci and are commonly present in the feces of warmblooded animals.
Enterococci are more persistent in water than coliforms. They provide a different assessment of the transport of fecal contamination in ground water than coliforms because of their different shape and survival rate.

Spore formers

Certain bacteria produce an environmentally resistant form called a spore.
Pathogens that produce spore-like structures include the protozoa, Cryptosporidium and Giardia.
Clostridium perfringens (C. perfringens), a spore-forming bacterium, is present in human and animal feces and may be useful as a surrogate for stress-resistant organisms.
C. perfringens is mainly an indicator of contamination from point sources, as well as a conservative tracer of past fecal contamination.

Bacterial Methods Bacteriological indicators are routinely measured by means of membrane-filtration or most-probable-number techniques.

Membrane-filtration technique		Most-probable-number technique-Colilert quantitray
Total coliforms and E. coli--MI and Colilert methods

MI method			Colilert quantitray method
Membrane-filtration method – allows simultaneous enumeration of total coliforms and E. coli. Medium contains a fluorogen that reacts with galactosidase in total coliforms, and a chromogen that reacts with glucoronidase in E. coli. Total coliforms fluoresce under ultraviolet light, and E. coli appear blue under ambient light, following a 24-hour incubation at 35 ºC.	Total coliforms on MI agar	E.coli on MI agar	Most-probable-number method–allows simultaneous enumeration of total coliforms and E. coli. Medium contains a chromogen that reacts with galactosidase in total coliforms, and a fluorogen that reacts with glucuronidase in E. coli. Total coliforms appear yellow under ambient light and E. coli fluoresce under ultraviolet light, following a 24-hour incubation at 35 ºC.

E. coli -mTEC and modified mTEC methods
mTEC method			Modified mTEC method
Two-step membrane-filtration method – allows detection of lactose fermentation and tests for the enzyme urease. E. coli colonies appear yellow, following a 2-hour incubation at 35 ºC and a 22-hour incubation at 44.5 ºC. Requires transfer of membrane to a urea-substrate medium – E. coli colonies are urease negative and will remain yellow.	E. coli on mTEC agar	E. coli on modified mTEC agar	One-step membrane-filtration method – allows detection of E. coli without the secondary test for urease activity. Medium contains a chromogen that reacts with the enzyme glucuronidase in E. coli. E. coli colonies appear magenta, following a 2-hour incubation at 35 ºC and a 22-hour incubation at 44.5 ºC.

Enterococci-mEI method

Clostridium perfringens-mCP method

Membrane-filtration method - allows detection of enterococci on a single medium, an improvement over the mE/EIA method.
Medium contains a chromagen that reacts with the enzyme glucosidase in enterococci.
Enterococci colonies have a blue halo, following a 24-hour incubation at 41 °C; colony color is unimportant.

Enterococci on
mEI agar

Membrane-filtration method - allows detection of the anaerobe C. perfringens by use of an oxygen-free chamber.
Medium contains a chromagen that reacts with the enzyme acid phosphatase in C. perfringens.
C. perfringens colonies turn magenta upon exposure to ammonium hydroxide vapors, following a 24-hour incubation under anaerobic conditions at 42 °C.

Clostridium perfringens
on mCP agar

Viral Indicators and Pathogens
Coliphage	Enteric viruses
Coliphage are viral indicators that infect and replicate in coliform bacteria, primarily E. coli. Coliphage are found in high numbers in sewage and may be better than bacterial indicators to represent the survival and transport of viruses in the environment. Two main groups of coliphage are detected in separate tests: Somatic coliphage infect the outer cell wall of its host and requires a laboratory strain, like E. coli CN-13, for detection. F-specific (also called male-specific) coliphage infect the F-pili (produced at temperatures above 25 ºC) of its host and requires a laboratory strain, like E. coli F-amp, for detection.	There are more than 100 different types of human pathogenic viruses that may be present in fecal-contaminated waters; however, only a small number can be detected by available methods. Viruses are generally more persistant in the environment than bacteria and may not be completely removed by treatment processes. Because of their smaller size, viruses are often transported further in ground water than bacteria or protozoa. Some of the more common viruses included in monitoring programs are enterovirus, hepatitis A virus, rotavirus, reovirus, and calicivirus.

Viral Methods
Coliphage – Single-agar layer (SAL) and two-step enrichment methods		Enteric viruses – reverse-transcriptase polymerase chain reaction (RT-PCR)
Documented and approved by U.S. Environmental Protection Agency (USEPA). Must be done by a trained analyst under sterile conditions. SAL method is a quantitative, 24-hour plaque assay method that is limited to sample volumes of 100 mL. Two-step enrichment method is a 48-hour presence/absence method that can be used to analyze sample volumes of either 100 mL or 1 L		Must be done in a laboratory designed for molecular work by a trained analyst. Amplifies and detects the genetic material of specific viruses from large volumes of water. Detects the presence of actual pathogens; however, does not determine the infectious state of the viruses.
Single-agar layer method	Two-step enrichment method	Detection of virus presence after RT-PCR

Protozoan Pathogens	Protozoan Method
Cryptosporidium and Giardia	Filtration/Immunomagnetic Separation (IMS)/ Immunofluorescence Assay (FA)
Cryptosporidium and Giardia are the principal protozoan pathogens that affect the public health acceptability of waters in the United States. Both protozoa are widely distributed in the aquatic environment and have been implicated in several waterborne-disease outbreaks. Both produce environmentally resistant forms (oocysts and cysts) that allow for their extended survival in water.	Documented and approved by USEPA to detect Cryptosporidium and Giardia from large volumes of water by filtration, IMS, and FA microscopy. Does not identify to the species level, nor does it determine the viability or infectivity of the detected organisms. Must be done in the laboratory by a trained analyst.

Cryptosporidium and Giardia	Immunomagnetic separation

Quality Assurance/Quality Control

The OWML is committed to providing quality microbiological analytical services to the USGS. The quality assurance/quality control (QA/QC) program is designed to ensure the production of scientifically sound, legally defensible data of known and documented quality. The QA/QC manual identifies and documents practices and standard operating procedures for the activities in the OWML that affect quality of data. The manual is frequently updated as laboratory activities expand and change.

Data Management

A laboratory information management system (LIMS) is used by the OWML to store sample login information, sample results, and associated quality-control results. This information is backed up daily, and a copy of the information is stored at an offsite location. The LIMS is used to store QA/QC records: maintenance and calibration of laboratory equipment, maintenance of microbiological stock cultures and controls, and laboratory method QA/QC results.

The LIMS has been customized to produce reports of results that can be easily uploaded into the USGS National Water Information System (NWIS). Once the results have been loaded into NWIS, the information can be retrieved through NWIS Web: http://water.usgs.gov/nwis. Currently, water-quality data are updated annually on NWIS Web.

Web Site

Contact information for the OWML staff can be found at the OWML Web site:
http://oh.water.usgs.gov/microbiol.html

Other information that can be found on the OWML Web site includes

information about microorganisms of public-health significance and the methods used to detect them
field sampling methods and hold times
current projects and publications
guidance for microbiology within the National Water-Quality Assessment (NAWQA) program
QA/QC manual
price list for internal analytical services and instructions for sample submission
references for methods described in this fact sheet

Additional Information

For additional information about the USGS and its programs, contact:

Director, U.S. Geological Survey, Ohio Water Science Center
6480 Doubletree Avenue, Columbus, OH 43229-1111
Email: dc_oh@usgs.gov
Phone: (614) 430-7700
Fax: (614) 430-7777

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the U.S. Government.

U.S. Department of the Interior
U.S. Geological Survey

Fact Sheet FS 2005–3072
June 2005

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For access to real-time and historical water data for Ohio, and for information about water-resource studies in the State, please visit our Web site at http://oh.water.usgs.gov/.

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Ohio Water Microbiology Laboratory

Overview

Mission

OWML laboratory technician analyzing a water sample for coliphage using a single-agar layer technique

OWML Capabilities

Bacterial Indicators

Coliforms

Streptococci

Spore formers

Bacterial Methods

Membrane-filtration technique

Most-probable-number technique-Colilert quantitray

Total coliforms and E. coli--MI and Colilert methods

MI method

Colilert quantitray method

Total coliforms on MI agar

E.coli on MI agar

E. coli -mTEC and modified mTEC methods

mTEC method

Modified mTEC method

E. coli on mTEC agar

E. coli on modified mTEC agar

Enterococci-mEI method

Clostridium perfringens-mCP method

Enterococci on mEI agar

Clostridium perfringens on mCP agar

Viral Indicators and Pathogens

Coliphage

Enteric viruses

Viral Methods

Coliphage – Single-agar layer (SAL) and two-step enrichment methods

Enteric viruses – reverse-transcriptase polymerase chain reaction (RT-PCR)

Single-agar layer method

Two-step enrichment method

Detection of virus presence after RT-PCR

Protozoan Pathogens

Protozoan Method

Cryptosporidium and Giardia

Filtration/Immunomagnetic Separation (IMS)/ Immunofluorescence Assay (FA)

Cryptosporidium and Giardia

Immunomagnetic separation

Quality Assurance/Quality Control

Data Management

Web Site

Additional Information

Most-probable-number
technique-Colilert quantitray

Total coliforms on
MI agar

E.coli on MI
agar

Enterococci on
mEI agar

Clostridium perfringens
on mCP agar

Coliphage – Single-agar layer (SAL) and two-step
enrichment methods

Detection of virus presence after
RT-PCR

Filtration/Immunomagnetic Separation (IMS)/
Immunofluorescence Assay (FA)