Ohio Water Microbiology Laboratory

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 publichealth significance in the environment.

• provide assistance to other USGS employees for project 

planning and training related to microorganisms of publichealth significance.

• continually develop projects and programs that enhance 

our understanding of the processes that affect microorganisms in the environment.

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 

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

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.

Fact Sheet FS 2005–3072

June 2005

U.S. Department of the Interior

U.S. Geological Survey Printed on recycled paper
Bacterial Indicators

• 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.

• 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.

• 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. 

Total coliforms and E. coli – MI and Colilert methods

MI 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.

Colilert quantitray method

 • 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

• 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.

Modified mTEC method

• 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.

Membrane-filtration technique Most-probable-number technique - Colilert quantitray

Total coliforms on E. coli on MI agar

MI agar

E. coli on mTEC agar

Coliforms

E. coli on modified 

mTEC agar

Streptococci Spore formers
Enterococci – mEI 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 oC; colony 

color is unimportant.

Clostridium perfringens - mCP method

• 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 oC. 

Viral Indicators and Pathogens

Coliphage

• 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:

o Somatic coliphage infect the outer cell wall of its host 

and requires a laboratory strain, like E. coli CN-13, for 

detection. 

o 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.

Enteric viruses

• 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 

• 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.

Enteric viruses – reverse-transcriptase polymerase chain 

reaction (RT-PCR)

• 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.

Enterococci on mEI agar Clostridium perfringens

on mCP agar

Single-agar layer method Two-step enrichment method Reproduced from Madigan and others, 2000

Detection of virus presence after 

RT-PCR
Cryptosporidium and Giardia

• 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 waterbornedisease outbreaks.

• Both produce environmentally resistant forms (oocysts and 

cysts) that allow for their extended survival in water.

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.

Cryptosporidium and Giardia Immunomagnetic separation H.D.A. Lindquist, USEPA

Filtration/Immunomagnetic Separation (IMS)/

Immunofluorescence Assay (FA) 

• 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. 

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.

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 

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 WaterQuality 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

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.

Protozoan Pathogens Protozoan Method