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Library of Congress Cataloging-in-Publication Data

Names: Yates, M. V. (Marylynn V.), editor. | Nakatsu, Cindy H., editor. | Miller, Robert V. (Robert Verne), 1945- editor. | Pillai, Suresh D., 1962- editor.

Title: Manual of environmental microbiology / editor in chief, Marylynn V. Yates; editors, Cindy H. Nakatsu, Robert V. Miller, Suresh D. Pillai.

Description: Fourth edition. | Washington, DC : ASM Press, [2016] | Includes bibliographical references and index.

Identifiers: LCCN 2016014816 (print) | LCCN 2016016986 (ebook) | ISBN 9781555816025 (hardcover) | ISBN 9781683673231 (ebook)

Subjects: LCSH: Microbial ecology–Laboratory manuals. | Sanitary microbiology–Laboratory manuals.

Classification: LCC QR100 .M36 2016 (print) | LCC QR100 (ebook) | DDC 577.8–dc23

LC record available at https://lccn.loc.gov/2016014816

All Rights Reserved

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CONTENTS

Editorial Board

Contributors

1.1.1Introduction

MARYLYNN V. YATES

GENERAL METHODOLOGY

VOLUME EDITOR: SURESH D. PILLAI

SECTION EDITORS: YOICHI KAMAGATA, CLEBER C. OUVERNEY, DOUGLAS R. CALL, STEFAN J. GREEN, YILDIZ T. CHAMBERS, AND JOHN SCOTT MESCHKE

2.1CULTURE-BASED AND PHYSIOLOGICAL DETECTION

2.1.1Detection of Specific Taxa Using Chromogenic and Fluorogenic Media

MOHAMMAD MANAFI

2.1.2Anaerobic Cultivation

TAKASHI NARIHIRO AND YOICHI KAMAGATA

2.1.3New Devices for Cultivation

YOSHITERU AOI AND SLAVA EPSTEIN

2.2MICROSCOPIC METHODS

2.2.1Gold-Based In Situ Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples

THILO EICKHORST AND HANNES SCHMIDT

2.2.2Assessment of Prokaryotic Biological Activity at the Single-Cell Level by Combining Microautoradiography and Fluorescence in situ Hybridization

CLEBER C. OUVERNEY

2.3TARGET-SPECIFIC DETECTION

2.3.1Antibody-Based Technologies for Environmental Biodetection

CHERYL L. BAIRD AND SUSAN M. VARNUM

2.3.2PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification

RACHEL A. BARTHOLOMEW, JANINE R. HUTCHISON, TIMOTHY M. STRAUB, AND DOUGLAS R. CALL

2.3.3Microarray-Based Environmental Diagnostics

DARRELL P. CHANDLER

2.3.4Field Application of Pathogen Detection Technologies

TIMOTHY M. STRAUB, DOUGLAS R. CALL, CINDY BRUCKNER-LEA, HEATHER COLBURN, CHERYL L. BAIRD, RACHEL A. BARTHOLOMEW, RICHARD OZANICH, AND KRISTIN JARMAN

2.4MICROBIAL COMMUNITY ANALYSIS OF ENVIRONMENTAL SAMPLES WITH NEXT-GENERATION SEQUENCING

2.4.1Introduction to Microbial Community Analysis of Environmental Samples with Next-Generation Sequencing

STEFAN J. GREEN AND JOSH D. NEUFELD

2.4.2Microbial Community Analysis Using High-Throughput Amplicon Sequencing

DANNY IONESCU, WILL A. OVERHOLT, MICHAEL D. J. LYNCH, JOSH D. NEUFELD, ANKUR NAQIB, AND STEFAN J. GREEN

2.4.3Functional Metagenomics: Procedures and Progress

LAURA S. MORRIS AND JULIAN R. MARCHESI

2.4.4Metagenomics: Assigning Functional Status to Community Gene Content

NASEER SANGWAN AND RUP LAL

2.4.5Generation and Analysis of Microbial Metatranscriptomes

NEHA SARODE, DARREN J. PARRIS, SANGITA GANESH, SHERRY L. SESTON, AND FRANK J. STEWART

2.5QA/QC IN ENVIRONMENTAL MICROBIOLOGY

2.5.1Introduction to Principles of Quality Assurance

KEVIN K. CONNELL

2.5.2General Quality Control

ROBIN K. OSHIRO

2.5.3Quality Control for Bacteriological Analyses

ELLEN BRAUN-HOWLAND

2.5.4Quality Control for Virological Analyses

RICHARD E. DANIELSON

2.5.5Quality Control for USEPA Method 1623 Protozoan Analysis and PCR Analyses

GEORGE D. DI GIOVANNI AND GREGORY D. STURBAUM

2.5.6The Role of Statistical Thinking in Environmental Microbiology

J. VAUN MCARTHUR AND R. CARY TUCKFIELD

2.5.7Study Design

YILDIZ T. CHAMBERS AND ROBIN K. OSHIRO

2.6SAMPLING METHODS

2.6.1Water Sampling and Processing Techniques for Public Health-Related Microbes

VINCENT HILL

2.6.2Surface Sampling

LAURA J. ROSE, JUDITH NOBLE-WANG, AND MATTHEW J. ARDUINO

2.6.3Soil Sampling for Microbial Analyses

JOHN BROOKS

2.6.4Microbiological Sampling of Wastewater and Biosolids

NICOLETTE A. ZHOU, ERIC C. THOMPSON, AND JOHN SCOTT MESCHKE

ENVIRONMENTAL PUBLIC HEALTH MICROBIOLOGY

VOLUME EDITOR: MARYLYNN V. YATES

SECTION EDITORS: GARY A. TORANZOS, MARK P. BUTTNER, ED TOPP, VALERIE J. HARWOOD, AND MARYLYNN V. YATES

3.1WATER

3.1.1Current and Developing Methods for the Detection of Microbial Indicators in Environmental Freshwaters and Drinking Waters

TASHA M. SANTIAGO-RODRIGUEZ, JULIE KINZELMAN, AND GARY A. TORANZOS

3.1.2Best Practices for Cyanobacterial Harmful Algal Bloom Monitoring

TIMOTHY G. OTTEN AND HANS W. PAERL

3.1.3Assessing the Efficiency of Wastewater Treatment

GRACIELA RAMÍREZ TORO AND HARVEY MINNIGH

3.1.4Epidemiologic Aspects of Waterborne Infectious Disease

SAMUEL DOREVITCH

3.1.5Waterborne Enteric Viruses: Diversity, Distribution, and Detection

MORTEZA ABBASZADEGAN AND ABSAR ALUM

3.1.6Detection of Protozoa in Surface and Finished Waters

ABSAR ALUM, ERIC N. VILLEGAS, SCOTT P. KEELY, KELLY R. BRIGHT, LAURA Y. SIFUENTES, AND MORTEZA ABBASZADEGAN

3.1.7Drinking Water Microbiology

MARYLYNN V. YATES

3.2AIR

3.2.1Introduction to Aerobiology

PAULA KRAUTER AND LINDA D. STETZENBACH

3.2.2Sampling for Airborne Microorganisms

SERGEY A. GRINSHPUN, MARK P. BUTTNER, GEDIMINAS MAINELIS, AND KLAUS WILLEKE

3.2.3Analysis of Bioaerosol Samples

PATRICIA CRUZ AND MARK P. BUTTNER

3.2.4Fate and Transport of Microorganisms in Air

GARY S. BROWN AND ALAN JEFF MOHR

3.2.5Airborne Fungi and Mycotoxins

DE-WEI LI, ECKARDT JOHANNING, AND CHIN S. YANG

3.2.6Airborne Bacteria, Archaea, and Endotoxin

PETER S. THORNE, CAROLINE DUCHAINE, AND PASCALE BLAIS LECOURS

3.2.7Airborne Viruses

SYED A. SATTAR, NITIN BHARDWAJ, AND M. KHALID IJAZ

3.2.8Aerobiology of Agricultural Pathogens

ESTELLE LEVETIN

3.2.9Legionellae and Legionnaires’ Disease

CLARESSA E. LUCAS AND BARRY S. FIELDS

3.3SOIL

3.3.1Pathogenic Viruses and Protozoa Transmitted by Soil

PASCAL DELAQUIS, JULIE BRASSARD, AND ALVIN GAJADHAR

3.3.2Natural Soil Reservoirs for Human Pathogenic and Fecal Indicator Bacteria

MARIA LAURA BOSCHIROLI, JOSEPH FALKINHAM, SABINE FAVRE-BONTÉ, SYLVIE NAZARET, PASCAL PIVETEAU, MICHAEL SADOWSKY, MURULEE BYAPPANAHALLI, PASCAL DELAQUIS, AND ALAIN HARTMANN

3.4MICROBIAL SOURCE TRACKING

3.4.1The Evolving Science of Microbial Source Tracking

VALERIE J. HARWOOD, CHARLES HAGEDORN, AND MICHAEL SADOWSKY

3.4.2Validation of Microbial Source Tracking Markers and Detection Protocols: Considerations for Effective Interpretation

ASJA KORAJKIC, DON STOECKEL, AND JOHN F. GRIFFITH

3.4.3Overview of Microbial Source Tracking Methods Targeting Human Fecal Pollution Sources

ORIN C. SHANKS, HYATT GREEN, ASJA KORAJKIC, AND KATHARINE G. FIELD

3.4.4Methods of Targeting Animal Sources of Fecal Pollution in Water

ANICET R. BLANCH, ELISENDA BALLESTÉ, JENNIFER WEIDHAAS, JORGE SANTO DOMINGO, AND HODON RYU

3.4.5Microbial Source Tracking: Field Study Planning and Implementation

JULIE KINZELMAN AND WARISH AHMED

3.4.6Fecal Indicator Organism Modeling and Microbial Source Tracking in Environmental Waters

MEREDITH B. NEVERS, MURULEEDHARA N. BYAPPANAHALLI, MANTHA S. PHANIKUMAR, AND RICHARD L. WHITMAN

3.5MICROBIAL RISK ASSESSMENT

3.5.1Risk Assessment Framework

MARYLYNN V. YATES

3.5.2Exposure Assessment

SUSAN R. PETTERSON AND NICHOLAS J. ASHBOLT

3.5.3Dose-Response Modeling and Use: Challenges and Uncertainties in Environmental Exposure

MARK H. WEIR

MICROBIAL ECOLOGY

VOLUME EDITOR: ROBERT V. MILLER

SECTION EDITORS: LARRY J. FORNEY, ROBERT H. FINDLAY, BRIAN P. HEDLUND, AND JULIAN R. MARCHESI

4.1THEORY

4.1.1Phylogenomic Networks of Microbial Genome Evolution

TAL DAGAN, OVIDIU POPA, THORSTEN KLÖSGES, AND GIDDY LANDAN

4.1.2Evolutionary Ecology of Microorganisms: From the Tamed to the Wild

JAY T. LENNON AND VINCENT J. DENEF

4.2AQUATIC ENVIRONMENTS

4.2.1The Microbial Ecology of Benthic Environments

ROBERT H. FINDLAY AND TOM J. BATTIN

4.2.2Heterotrophic Planktonic Microbes: Viruses, Bacteria, Archaea, and Protozoa

JED A. FUHRMAN AND DAVID A. CARON

4.2.3Aquatic Biofilms: Development, Cultivation, Analyses, and Applications

JOHN R. LAWRENCE, THOMAS R. NEU, ARMELLE PAULE, DARREN R. KORBER, AND GIDEON M. WOLFAARDT

4.3EXTREME ENVIRONMENTS

4.3.1The Microbiology of Extremely Acidic Environments

D. BARRIE JOHNSON AND ANGELES AGUILERA

4.3.2Life in High Salinity Environments

AHARON OREN

4.3.3Microbial Life in Extreme Low-Biomass Environments: A Molecular Approach

KASTHURI VENKATESWARAN, MYRON T. LA DUC, PARAG VAISHAMPAYAN, AND JAMES A. SPRY

4.3.4Life in High-Temperature Environments

BRIAN P. HEDLUND, SCOTT C. THOMAS, JEREMY A. DODSWORTH, AND CHUANLUN L. ZHANG

4.4ANIMAL-GUT MICROBIOMES

4.4.1Invertebrate Gut Associations

DANIELE DAFFONCHIO, ALBERTO ALMA, GUIDO FAVIA, LUCIANO SACCHI, AND CLAUDIO BANDI

4.4.2Studying the Mammalian Intestinal Microbiome Using Animal Models

FLOOR HUGENHOLTZ, JING ZHANG, PAUL W. O’TOOLE, AND HAUKE SMIDT

4.4.3Animal Gut Microbiomes

RICHARD J. ELLIS AND CHRISTOPHER S. MCSWEENEY

BIODEGRADATION AND BIOTRANSFORMATION

VOLUME EDITOR: CINDY H. NAKATSU

SECTION EDITORS: CINDY H. NAKATSU AND CHRISTOPHER RENSING

5.1BIODEGRADATION

5.1.1Genomic Features and Genome-Wide Analysis of Dioxin-Like Compound Degraders

MASAKI SHINTANI AND KAZUHIDE KIMBARA

5.1.2Biodegradation of Organochlorine Pesticides

YUJI NAGATA, MICHIRO TABATA, YOSHIYUKI OHTSUBO, AND MASATAKA TSUDA

5.1.3Anaerobic Degradation of Aromatic Compounds

WEIMIN SUN, VALDIS KRUMINS, DONNA E. FENNELL, LEE J. KERKHOF, AND MAX M. HÄGGBLOM

5.1.4Microbial Electrochemical Technologies Producing Electricity and Valuable Chemicals from Biodegradation of Waste Organic Matters

TAEHO LEE, AKIHIRO OKAMOTO, SOKHEE JUNG, RYUHEI NAKAMURA, JUNG RAE KIM, KAZUYA WATANABE, AND KAZUHITO HASHIMOTO

5.1.5A Basic Introduction to Aerobic Biodegradation of Petroleum Aromatic Compounds

KENGO INOUE, ONRUTHAI PINYAKONG, KANO KASUGA, AND HIDEAKI NOJIRI

5.1.6Environmental Systems Microbiology of Contaminated Environments

TERRY C. HAZEN AND GARY S. SAYLER

5.2BIOTRANSFORMATION

5.2.1Breathing Iron: Molecular Mechanism of Microbial Iron Reduction by Shewanella oneidensis

REBECCA E. COOPER, JENNIFER L. GOFF, BEN C. REED, RAMANAN SEKAR, AND THOMAS J. DICHRISTINA

5.2.2Experimental Geomicrobiology: From Field to Laboratory

TIMOTHY S. MAGNUSON AND RHESA N. LEDBETTER

5.2.3Microbial Uses in the Remediation of Metal-Impacted Soils

TIMBERLEY ROANE AND MUNIRA LANTZ

Index

EDITORIAL BOARD

Mark P. ButtnerSection 3.2

School of Community Health Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154

Douglas R. CallSection 2.3

Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164

Yildiz T. ChambersSection 2.5

CSC Science, Engineering, and Mission Support, Alexandria, VA 22310

Robert H. FindlaySection 4.2

University of Alabama, Department of Biological Sciences, Tuscaloosa, AL 35487

Larry J. ForneySection 4.1

Department of Biological Sciences, University of Idaho, Moscow, ID 83844

Stefan J. GreenSection 2.4

Research Resources Center, University of Illinois at Chicago, Chicago, IL 60612

Valerie J. HarwoodSection 3.4

Department of Integrative Biology, University of South Florida, Tampa, FL 33620

Brian P. HedlundSection 4.3

School of Life Sciences, Nevada Institute of Personalized Medicine, University of Nevada, Las Vegas, Las Vegas, NV 89154

Yoichi KamagataSection 2.1

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan

Julian R. MarchesiSection 4.4

School of Biosciences, Cardiff University, Centre for Digestive and Gut Health, Imperial College London, Cardiff, Wales CF10 3AT, United Kingdom

John Scott MeschkeSection 2.6

Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105

Cleber C. OuverneySection 2.2

Department of Biological Sciences, San Jose State University, San Jose, CA 95192

Christopher RensingSection 5.2

Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg 1871, Denmark

Ed ToppSection 3.3

Agriculture and Agri-Food Canada, London, ON N5V 4T3, Canada

Gary A. ToranzosSection 3.1

Department of Biology, University of Puerto Rico, San Juan, PR 00932, Puerto Rico

CONTRIBUTORS

MORTEZA ABBASZADEGAN

Arizona State University, Tempe, AZ 85287

ANGELES AGUILERA

Centro de Astrobiología (INTA-CSIC), Madrid 28850, Spain

WARISH AHMED

CSIRO Land and Water Queensland Biosciences Precinct, St. Lucia, Queensland 4067, Australia

ALBERTO ALMA

Department of Agriculture, Forestry and Food Sciences DISAFA, University of Turin, Grugliasco I-10095, Italy

ABSAR ALUM

Arizona State University, Tempe, AZ, 85287

YOSHITERU AOI

Institute of Sustainable Sciences and Development, Hiroshima University, Hiroshima 739-8529 Japan, and Northeastern University, Boston, MA 02115

MATTHEW J. ARDUINO

Centers for Disease Control and Prevention, Division of Healthcare Quality Promotion, Atlanta, GA 30329

NICHOLAS J. ASHBOLT

School of Public Health, University of Alberta, Edmonton, AB T6G 2G7, Canada

CHERYL L. BAIRD

Pacific Northwest National Laboratory, Biochemistry and Structural Biology, Fundamental and Computational Sciences Division, Richland, WA 99352

ELISENDA BALLESTÉ

Department of Microbiology, University of Barcelona, Barcelona 08028, Spain

CLAUDIO BANDI

Department of Veterinary Sciences and Public Health, University of Milan, Milan I-20133, Italy

RACHEL A. BARTHOLOMEW

Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, Richland, WA 99354

TOM J. BATTIN

Stream Biofilm and Ecosystem Research Laboratory, Ecole Polytechnique Fédérale Lausanne, CH-1015 Lausanne, Switzerland

NITIN BHARDWAJ

Advanced Medical Research Institute of Canada, Sudbury, ON P3E 5J1, Canada

PASCALE BLAIS LECOURS

Centre deRecherché, University Institute of Cardiology and Pulmonology of Québec, Université of Laval, Québec, QC G1K7P4, Canada

ANICET R. BLANCH

Department of Microbiology, University of Barcelona, Barcelona 08028, Spain

MARIA LAURA BOSCHIROLI

ANSES French Agency for Food, Environmental & Occupational Health Safety, Maisons-Alfort Animal Health Laboratory, Bacterial Zoonoses Unit, Maisons-Alfort 94706, France

JULIE BRASSARD

Agriculture and Agri-Food Canada, Food Research and Development Centre, Saint-Hyacinthe, QC J2S 8E3, Canada

ELLEN BRAUN-HOWLAND

Laboratory of Environmental Biology, NYSDOH, Wadsworth Center, Biggs Laboratory, Empire State Plaza , Albany, NY 12201

KELLY R. BRIGHT

University of Arizona, Tempe, AZ 85287

JOHN BROOKS

Genetics and Precision Agriculture Unit, USDA-ARS, Mississippi State University, Mississippi State, MS 39762

GARY S. BROWN

Lockheed Martin Corporation, Scientific, Engineering, Response, Analytical Services, Las Vegas, NV 89119

CINDY BRUCKNER-LEA

Pacific Northwest National Laboratory, Richland, WA 99354

MARK P. BUTTNER

School of Community Health Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154

MURULEE BYAPPANAHALLI

USGS Great Lakes Science Center, Ann Arbor, MI 48105

DOUGLAS R. CALL

Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164

DAVID A. CARON

Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089

YILDIZ T. CHAMBERS

CSC Science, Engineering, and Mission Support, Alexandria, VA 22310

DARRELL P. CHANDLER

Akonni Biosystems, Inc., Frederick, MD 21701

HEATHER COLBURN

Pacific Northwest National Laboratory, Richland, WA 99354

KEVIN K. CONNELL

Science & Engineering Line of Service, CSC, Alexandria, VA 22310

REBECCA E. COOPER

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

PATRICIA CRUZ

School of Community Health Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154

DANIELE DAFFONCHIO

Department of Food, Environmental and Nutritional, Sciences, DeFENS, University of Milan, Milan I-20133, Italy

TAL DAGAN

Institute of Microbiology, Christian-Albrechts-University of Kiel, Kiel 24118, Germany

RICHARD E. DANIELSON

BioVir Laboratories, Inc., Benicia, CA 94510

PASCAL DELAQUIS

Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BC V0H 1Z0, Canada

VINCENT J. DENEF

Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109

GEORGE D. DI GIOVANNI

Environmental and Occupational Health Sciences, University of Texas School of Public Health, El Paso Regional Campus, El Paso, TX 79902

THOMAS J. DICHRISTINA

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

JEREMY A. DODSWORTH

Department of Biology, California State University, San Bernardino, CA 92407

SAMUEL DOREVITCH

U of Illinois at Chicago, School of Public Health, Chicago, IL 60612

CAROLINE DUCHAINE

Department of Biochemistry and Microbiology, Université Laval, Québec, QC G1K7P4, Canada

THILO EICKHORST

Soil Microbial Ecology, University of Bremen, Bremen 28359, Germany

RICHARD J. ELLIS

Animal and Plant Health Agency, Specialist Scientific Support Department, New Haw, Surrey KT15 3NB, United Kingdom

SLAVA EPSTEIN

Department of Biology, Northeastern University, Boston, MA 02115

JOSEPH FALKINHAM

III, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061

GUIDO FAVIA

School of Biosciences and Biotechnology, University of Camerino, Camerino I-62032, Italy

SABINE FAVRE-BONTÉ

Microbial Ecology Laboratory, UMR 5557, CNRS/University Lyon I, Villeurbanne 69622, France

DONNA E. FENNELL

Department of Environmental Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

KATHARINE G. FIELD

Oregon State University, Department of Microbiology, Corvallis, OR 97331

BARRY S. FIELDS

Division of Global Disease Detection & Emergency Response, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, GA 30333

ROBERT H. FINDLAY

University of Alabama, Department of Biological Sciences, Tuscaloosa, AL 35487

JED A. FUHRMAN

Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089

ALVIN GAJADHAR

Canadian Food Inspection Agency, Centre for Foodborne and Animal Parasitology, Saskatoon, SK S7N 2R3, Canada

SANGITA GANESH

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

JENNIFER L. GOFF

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

HYATT GREEN

US EPA, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, OH 45268

STEFAN J. GREEN

Research Resources Center, University of Illinois at Chicago, Chicago, IL 60612

JOHN F. GRIFFITH

Southern California Coastal Water Research Program, Costa Mesa, CA 92626

SERGEY A. GRINSHPUN

University of Cincinnati, Center for Health-Related Aerosol Studies, Cincinnati, OH 45267

CHARLES HAGEDORN

Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061

MAX M. HÄGGBLOM

Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

ALAIN HARTMANN

Agroecology Unit, UMR 1347, INRA/University of Burgundy/AgroSup Dijon, Dijon 21065, France

VALERIE J. HARWOOD

Department of Integrative Biology, University of South Florida, Tampa, FL 33620

KAZUHITO HASHIMOTO

Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan

TERRY C. HAZEN

Department of Civil & Environmental Engineering, University of Tennessee/Oak Ridge National Laboratory, Knoxville, TN 37996

BRIAN P. HEDLUND

School of Life Sciences, Nevada Institute of Personalized Medicine, University of Nevada, Las Vegas, Las Vegas, NV 89154

VINCENT HILL

Centers for Disease Control and Prevention, Atlanta, GA 30333

FLOOR HUGENHOLTZ

Wageningen University, Laboratory of Microbiology, TI Food and Nutrition, Netherlands Consortium for Systems Biology, University of Amsterdam, Wageningen 6703HB, The Netherlands

JANINE R. HUTCHISON

Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, Richland, WA 99354

M. KHALID IJAZ

R&D Surface Care and Germ Protection, Reckitt Benckiser LLC., Montvale, NJ 07645

KENGO INOUE

University of Miyazaki, Biochemistry and Applied Biosciences, Miyazaki 889-2192, Japan

DANNY IONESCU

Leibniz Institute for Freshwater Ecology and Inland Fisheries, Neuglobsow, Stechlin 16775, Germany

KRISTIN JARMAN

Pacific Northwest National Laboratory, Applied Statisticals and Computational Modeling Group, Fundamental and Computational Sciences Directorate, Richland, WA 99354

ECKARDT JOHANNING

Occupational & Environmental Life Science, Fungal Research Group Foundation, Inc., Albany, NY 12203

D. BARRIE JOHNSON

College of Natural Sciences, Bangor University, Bangor LL57 2UW, United Kingdom

SOKHEE JUNG

School of Civil and Environmental Engineering, Yonsei University, Seoul 120-749, Korea

YOICHI KAMAGATA

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan

KANO KASUGA

Akita Prefectural University, Department of Biotechnology, Akita 010-0195, Japan

SCOTT P. KEELY

United States Environmental Protection Agency, Cincinnati, OH 45268

LEE J. KERKHOF

Department of Marine and Coastal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

JUNG RAE KIM

School of Chemical and Biomolecular Engineering, Pusan National University, Pusan 609-735, Korea

KAZUHIDE KIMBARA

Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University, Hamamatsu, Shizuoka 432-8561, Japan

JULIE KINZELMAN

City of Racine Health Department, Racine, WI 53403

THORSTEN KLÖSGES

Institute of Molecular Evolution, Heinrich-Heine University Düsseldorf, Düsseldorf 40225, Germany

ASJA KORAJKIC

US Environmental Protection Agency, Cincinnati, OH 45268

DARREN R. KORBER

Department of Food and Bioproduct Science, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada

PAULA KRAUTER

Sandia National Laboratories (retired), Livermore, CA 94550

VALDIS KRUMINS

Department of Environmental Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

MYRON T. LA DUC

Jet Propulsion Lab, California Institute of Technology, Pasadena, CA 91109

RUP LAL

Department of Zoology, University of Delhi, Delhi 110007, India

GIDDY LANDAN

Institute of Microbiology, Christian-Albrechts-University of Kiel, Kiel 24118, Germany

MUNIRA LANTZ

Department of Integrative Biology, University of Colorado, Denver, Denver, CO 80217

JOHN R. LAWRENCE

Environment Canada, Saskatoon, SK S7N3H5, Canada

RHESA N. LEDBETTER

Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322

TAEHO LEE

Department of Environmental Engineering, Pusan National University, Pusan 609-735, Korea

JAY T. LENNON

Department of Biology, Indiana University, Bloomington, IN 47405

ESTELLE LEVETIN

Department of Biological Science, The University of Tulsa, Tulsa, OK 74104

DE-WEI LI

Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095

CLARESSA E. LUCAS

Division of Bacterial Diseases, National Center for Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333

MICHAEL D.J. LYNCH

Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada

TIMOTHY S. MAGNUSON

Department of Biological Sciences, Idaho State University, Pocatello, ID 83209

GEDIMINAS MAINELIS

Rutgers University, New Brunswick, NJ 08901

MOHAMMAD MANAFI

Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna 1090, Austria

JULIAN R. MARCHESI

School of Biosciences, Cardiff University, Centre for Digestive and Gut Health, Imperial College London, Cardiff, Wales CF10 3AT, United Kingdom

J. VAUN MCARTHUR

Savannah River Ecology Laboratory, Aiken, SC 29803

CHRISTOPHER S. MCSWEENEY

CSIRO Animal, Food and Health Services, Queensland Biosciences Precinct, St. Lucia, Queensland 4067, Australia

JOHN SCOTT MESCHKE

Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105

HARVEY MINNIGH

Gabriella and Paul Rosenbaum Foundation, Bryn Mawr, PA 19010

ALAN JEFF MOHR

Life Sciences Division, U.S. Army, Dugway Proving Ground, Dugway, UT 84022

LAURA S. MORRIS

School of Biosciences, Cardiff University, Cardiff, Wales CF10 3AT, United Kingdom

YUJI NAGATA

Department of Environmental Life Sciences, Graduate School of Life Sciences Tohoku University, Hatahira, Sendai 980-8577, Japan

RYUHEI NAKAMURA

Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan

ANKUR NAQIB

DNA Services Facility, University of Illinois at Chicago, Chicago, IL, 60613

TAKASHI NARIHIRO

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba 605-8566, Japan, and Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801

SYLVIE NAZARET

Microbial Ecology Laboratory, UMR 5557, CNRS/University Lyon I, Villeurbanne 69622, France

THOMAS R. NEU

River Ecology, Helmholtz Centre for Environmental Research, Magdeburg 39114, Germany

JOSH D. NEUFELD

Department of Biology, University of Waterloo, Waterloo, ON NSL 3G1, Canada

MEREDITH B. NEVERS

U.S. Geological Survey, Great Lakes Science Center, Porter, IN 46304

JUDITH NOBLE-WANG

Centers for Disease Control and Prevention, Division of Healthcare Quality Promotion, Atlanta, GA 30329

HIDEAKI NOJIRI

The University of Tokyo, Biotechnology Research Center, Tokyo 13-8657, Japan1

YOSHIYUKI OHTSUBO

Department of Environmental Life Sciences, Graduate School of Life Sciences Tohoku University, Hatahira, Sendai 980-8577, Japan

AKIHIRO OKAMOTO

Department of Earth Sciences, University of Southern California, Los Angeles, CA 90089

AHARON OREN

Department of Plant and Environmental Sciences, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

ROBIN K. OSHIRO

Engineering and Analysis Division, USEPA Headquarters, Washington, DC 20460

PAUL W. O’TOOLE

School of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Cork T12 YN60, Ireland

TIMOTHY G. OTTEN

Department of Microbiology, Oregon State University, Corvallis, OR 97331

CLEBER C. OUVERNEY

Department of Biological Sciences, San Jose State University, San Jose, CA 95192

WILL A. OVERHOLT

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

RICHARD OZANICH

Pacific Northwest National Laboratory, Richland, WA 99355

HANS W. PAERL

Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, NC 28557

DARREN J. PARRIS

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

ARMELLE PAULE

Global Institute for Water Security, Saskatoon, SK S7N3H5, Canada

SUSAN R. PETTERSON

Water & Health Pty Ltd, Salamander Bay, NSW 2317, Australia

MANTHA S. PHANIKUMAR

Michigan State University, Department of Civil and Environmental Engineering, East Lansing, MI 48824

ONRUTHAI PINYAKONG

Chulalongkorn University, Department of Microbiology, Bangkok 10330, Thailand

PASCAL PIVETEAU

Agroecology Unit, UMR 1347 INRA/University of Burgundy/AgroSup Dijon, Dijon 21065, France

OVIDIU POPA

Institute of Microbiology, Christian-Albrechts-University of Kiel, Kiel 24118, Germany

GRACIELA RAMÍREZ TORO

Centro de Educación, Conservación e Interpretación Ambiental, Universidad Interamericana de Puerto Rico, San Germán, PR 00683, Puerto Rico

BEN C. REED

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

TIMBERLEY ROANE

Department of Integrative Biology, University of Colorado, Denver, Denver, CO 80217

LAURA J. ROSE

Centers for Disease Control and Prevention, Division of Healthcare Quality Promotion, Atlanta, GA 30329

HODON RYU

US EPA NRMRL/WSWRD/MCCB, Cincinnati, OH 45268

LUCIANO SACCHI

Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia I-27100, Italy

MICHAEL SADOWSKY

BioTechnology Institute, University of Minnesota, St. Paul, MN 55108

NASEER SANGWAN

Department of Zoology, University of Delhi, Delhi 110007, India

TASHA M. SANTIAGO-RODRIGUEZ

Department of Biology, Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo, CA 93407

JORGE SANTO DOMINGO

US EPA NRMRL/WSWRD/MCCB, Cincinnati, OH 45268

NEHA SARODE

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

SYED A. SATTAR

Centre for Research on Environmental Microbiology, University of Ottawa, Ottawa, ON K1H 8M5, Canada

GARY S. SAYLER

Department of Microbiology, University of Tennessee, Knoxville, TN 37996

HANNES SCHMIDT

Soil Microbial Ecology, University of Bremen, Bremen 28359, Germany

RAMANAN SEKAR

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

SHERRY L. SESTON

Department of Biology, Alverno College, Milwaukee, WI 53234

ORIN C. SHANKS

US EPA, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, OH 45268

MASAKI SHINTANI

Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University, Hamamatsu, Shizuoka 432-8561, Japan

LAURA Y. SIFUENTES

University of Arizona, Tempe, AZ 85287

HAUKE SMIDT

Wageningen University, Laboratory of Microbiology, TI Food and Nutrition, Netherlands Consortium for Systems Biology, University of Amsterdam, Wageningen 6703HB, The Netherlands

JAMES A. SPRY

Jet Propulsion Lab, California Institute of Technology, Pasadena, CA 91109

LINDA D. STETZENBACH

University of Nevada, Las Vegas, Las Vegas, NV 89154

FRANK J. STEWART

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

DON STOECKEL

Batelle Memorial Institute, Columbus, OH 43201

TIMOTHY M. STRAUB

Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, Richland, WA 99354

GREGORY D. STURBAUM

ALS Laboratory Group, Molecular Biology, Scoresby, VIC 3179, Australia

WEIMIN SUN

Department of Biochemistry and Microbiology, Department of Environmental Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

MICHIRO TABATA

Department of Environmental Life Sciences, Graduate School of Life Sciences Tohoku University, Hatahira, Sendai 980-8577, Japan

SCOTT C. THOMAS

School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154

ERIC C. THOMPSON

King County Environmental Laboratory, Seattle, WA 98119

PETER S. THORNE

Department of Occupational and Environmental Health, The University of Iowa, College of Public Health, Iowa City, IA 52246

GARY A. TORANZOS

Department of Biology, University of Puerto Rico, San Juan, PR 00932, Puerto Rico

MASATAKA TSUDA

Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Hatahira, Sendai 980-8577, Japan

R. CARY TUCKFIELD

ECOSTATys LLC., Aiken, SC 29803

PARAG VAISHAMPAYAN

Jet Propulsion Lab, California Institute of Technology, Pasadena, CA 91109

SUSAN M. VARNUM

Pacific Northwest National Laboratory, Biochemistry and Structural Biology, Fundamental and Computational Sciences Division, Richland, WA 99352

KASTHURI VENKATESWARAN

Jet Propulsion Lab, California Institute of Technology, Pasadena, CA 91109

ERIC N. VILLEGAS

United States Environmental Protection Agency, Cincinnati, OH 45268

KAZUYA WATANABE

School of Life Science, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan

JENNIFER WEIDHAAS

West Virginia University, Civil and Environmental Engineering, Morgantown, WV 26506

MARK H. WEIR

Department of Public Health, Department of Civil and Environmental Engineering, Temple University, Philadelphia, PA 19122

RICHARD L. WHITMAN

U.S. Geological Survey, Great Lakes Science Center, Porter, IN 46304

KLAUS WILLEKE

University of Cincinnati, Cincinnati, OH 45267

GIDEON M. WOLFAARDT

Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B2K3, Canada

CHIN S. YANG

Prestige EnviroMicrobiology, Voorhees, NJ 08043

MARYLYNN V. YATES

Department of Environmental Sciences, University of California, Riverside, Riverside, CA 92521

CHUANLUN L. ZHANG

State Key Laboratory of Marine Geology School of Ocean and Earth Sciences, Tongji University, Shanghai 201804, China

JING ZHANG

Wageningen University, Laboratory of Microbiology, Wageningen 6703HB, The Netherlands

NICOLETTE A. ZHOU

Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105

INTRODUCTION

MARYLYNN V. YATES

1.1.1

Environmental microbiology might be considered by some to be an ill-defined subject: Where does the environment begin, and where does it end? From Marcus Terentius Varro’s observations regarding unseen “minute creatures” more than two millennia ago to Antonie van Leeuwenhoek’s first glimpse of the “animalcula” beneath his lens, there is no place on Earth—from thermophilic, acidic springs to the air we breathe to the deepest subsurface locations we have yet been able to reach—where people have looked and not found microorganisms of some type. The domain of what may be considered environmental microbiology thus continues to expand beyond the textbook definition of “the study of microorganisms existing in natural and artificial environments.” At the same time, our knowledge of microorganisms is increasing at an ever-more rapid rate as the result of incredible improvements in analytical methodology, especially at the molecular level. When compiling a manual of this nature, therefore, how does one determine what to include and what to exclude? In the end, the editors decided to showcase as much information as possible on some of the most important areas of environmental microbiology, to provide a clear sense of the possibilities presented by the existence of microorganisms in various environments. Further and more detailed information can be found in the wealth of expertly chosen references within each chapter.

This edition of the Manual of Environmental Microbiology has been reorganized, with new and updated sections to provide recent information on topics of importance to the field. The General Methodology volume, beginning with Section 2.1, is devoted to methodologies—the “how” of environmental microbial studies from analytical detection to sample collection. The methods presented in this volume are the basis of how we understand the microbial world around us and are used across environmental microbial disciplines and applications. General Methodologies begins with tried-and-true culture-based and physiological detection methods, microscopy-based methods, and molecular target–specific detection methods, before moving on to address the increasingly important study of microbes at the community level, thanks to the advent of next-generation sequencing technologies. This volume concludes with information on critical topics that are sometimes overlooked in environmental microbiology: quality assurance and quality control, which increase the usefulness and reliability of analytical data for downstream decision making.

The next volume, Environmental Public Health Microbiology, surveys the microorganisms in the water, air, or soil that can cause illness when humans are exposed to them. These potentially pathogenic microorganisms can take many avenues en route to their target host, ultimately leading to substantial health impacts and considerable negative economic and social consequences to the communities involved. The importance of understanding microbial distribution and detection for public health cannot be understated. This volume includes chapters describing the field of microbial source tracking; methods and approaches for determining predominant sources of fecal pollution of water, which has become a critical component of assessment; and determining ways to better protect water from microbial contamination. It culminates in a section on microbial risk assessment, integrating the information in the earlier sections and providing perspective for informed decision making and rational project design.

The Microbial Ecology volume addresses the various ways microorganisms can be classified and their interconnected relationships, with each other and with macroorganisms, and impacts on the environment. Some microorganisms are native to their environment and perform essential services, such as cycling nutrients or interacting with other organisms in ways that enable them to perform functions and/or inhabit environments where they would otherwise not exist. Other microorganisms may be introduced to a new environment, changing the overall ecosystem. This volume begins with an exploration of the theories behind microbial genome evolution and the evolutionary ecology of microorganisms. The subject matter then delves into more specific environments, including aquatic environments, environments with extreme conditions (extreme acidity, high salinity, low biomass, and high temperatures), and the unique environment inside the gastrointestinal tract of animals, an area of increasing study and data.

The final volume, Biodegradation and Biotransformation, focuses on the varied ways that microorganisms can transform or degrade nearly any natural or anthropogenic chemical compound. Since the earliest eras of the Industrial Revolution, study of microorganisms for these processes has been an active area of research. Microbial transformations of natural substances can make otherwise unavailable nutrients accessible by living organisms. Additionally, microorganisms can be used to degrade harmful contaminants, either by using the biochemical pathways naturally present or by employing genetic modification. These abilities have been exploited in numerous situations in which soil or water has been contaminated by organic or inorganic chemicals as well as for a variety of industrial and commercial uses.

Microorganisms play a critical role in the health and well-being of the planet and the human, animal, and plant life that dwells here. Our understanding of them is crucial for maintaining the environment. This fourth edition of the Manual of Environmental Microbiology builds on the solid foundation created by the previous three editions, and we are indebted to those who came before us. This edition is truly a collaborative triumph, and is the work of 19 editors and more than 180 contributing authors who generously gave their time, effort, and expertise. It is my hope that this reference serves as an informative and reliable source of information for current and future endeavors in environmental microbiology and inspires the next generation of researchers as they move along their path in this growing and important area.

GENERAL METHODOLOGY

VOLUME EDITOR: SURESH D. PILLAI

SECTION EDITORS: YOICHI KAMAGATA, CLEBER C. OUVERNEY, DOUGLAS R. CALL, STEFAN J. GREEN, YILDIZ T. CHAMBERS, AND JOHN SCOTT MESCHKE

2.1CULTURE-BASED AND PHYSIOLOGICAL DETECTION

2.1.1Detection of Specific Taxa Using Chromogenic and Fluorogenic Media

MOHAMMAD MANAFI

2.1.2Anaerobic Cultivation

TAKASHI NARIHIRO AND YOICHI KAMAGATA

2.1.3New Devices for Cultivation

YOSHITERU AOI AND SLAVA EPSTEIN

2.2MICROSCOPIC METHODS

2.2.1Gold-Based In Situ Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples

THILO EICKHORST AND HANNES SCHMIDT

2.2.2Assessment of Prokaryotic Biological Activity at the Single-Cell Level by Combining Microautoradiography and Fluorescence in situ Hybridization

CLEBER C. OUVERNEY

2.3TARGET-SPECIFIC DETECTION

2.3.1Antibody-Based Technologies for Environmental Biodetection

CHERYL L. BAIRD AND SUSAN M. VARNUM

2.3.2PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification

RACHEL A. BARTHOLOMEW, JANINE R. HUTCHISON, TIMOTHY M. STRAUB, AND DOUGLAS R. CALL

2.3.3Microarray-Based Environmental Diagnostics

DARRELL P. CHANDLER

2.3.4Field Application of Pathogen Detection Technologies

TIMOTHY M. STRAUB, DOUGLAS R. CALL, CINDY BRUCKNER-LEA, HEATHER COLBURN, CHERYL L. BAIRD, RACHEL A. BARTHOLOMEW, RICHARD OZANICH, AND KRISTIN JARMAN

2.4MICROBIAL COMMUNITY ANALYSIS OF ENVIRONMENTAL SAMPLES WITH NEXT-GENERATION SEQUENCING

2.4.1Introduction to Microbial Community Analysis of Environmental Samples with Next-Generation Sequencing

STEFAN J. GREEN AND JOSH D. NEUFELD

2.4.2Microbial Community Analysis Using High-Throughput Amplicon Sequencing

DANNY IONESCU, WILL A. OVERHOLT, MICHAEL D. J. LYNCH, JOSH D. NEUFELD, ANKUR NAQIB, AND STEFAN J. GREEN

2.4.3Functional Metagenomics: Procedures and Progress

LAURA S. MORRIS AND JULIAN R. MARCHESI

2.4.4Metagenomics: Assigning Functional Status to Community Gene Content

NASEER SANGWAN AND RUP LAL

2.4.5Generation and Analysis of Microbial Metatranscriptomes

NEHA SARODE, DARREN J. PARRIS, SANGITA GANESH, SHERRY L. SESTON, AND FRANK J. STEWART

2.5QA/QC IN ENVIRONMENTAL MICROBIOLOGY

2.5.1Introduction to Principles of Quality Assurance

KEVIN K. CONNELL

2.5.2General Quality Control

ROBIN K. OSHIRO

2.5.3Quality Control for Bacteriological Analyses

ELLEN BRAUN-HOWLAND

2.5.4Quality Control for Virological Analyses

RICHARD E. DANIELSON

2.5.5Quality Control for USEPA Method 1623 Protozoan Analysis and PCR Analyses

GEORGE D. DI GIOVANNI AND GREGORY D. STURBAUM

2.5.6The Role of Statistical Thinking in Environmental Microbiology

J. VAUN MCARTHUR AND R. CARY TUCKFIELD

2.5.7Study Design

YILDIZ T. CHAMBERS AND ROBIN K. OSHIRO

2.6SAMPLING METHODS

2.6.1Water Sampling and Processing Techniques for Public Health-Related Microbes

VINCENT HILL

2.6.2Surface Sampling

LAURA J. ROSE, JUDITH NOBLE-WANG, AND MATTHEW J. ARDUINO

2.6.3Soil Sampling for Microbial Analyses

JOHN BROOKS

2.6.4Microbiological Sampling of Wastewater and Biosolids

NICOLETTE A. ZHOU, ERIC C. THOMPSON, AND JOHN SCOTT MESCHKE

Detection of Specific Taxa Using Chromogenic and Fluorogenic Media

MOHAMMAD MANAFI

2.1.1

OVERVIEW OF FLUOROGENIC AND CHROMOGENIC MEDIA IN ENVIRONMENTAL MICROBIOLOGY

Over the past 20 years, a number of selective chromogenic and fluorogenic media for detection and enumeration of most important bacteria and yeast in particular in food and water have been developed and marketed. Fluorogenic enzyme substrates generally consist of a specific substrate for the specific enzyme such as sugar or amino acid and a fluorogen such as 4-methylumbelliferone, being able to convert UV light (365 nm) to visible light. Chromogenic enzyme substrates are compounds that act as the substrate for specific enzymes and change color due to the action of the enzymes. Most commercially available chromogenic media have exploited indoxylic substrate. Indoxyl and its halogenated derivatives can be derivatized to form a range of esters. Halogenation of the indoxyl molecule has an effect on the color and intensity of the chromogen. 5-Bromo-4-chloro-indoxyl forms a green/blue dye, whereas 5-bromo-6-chloro-indoxyl forms a magenta dye. The incorporation of such substrates into a selective medium can eliminate the need for subculture and further biochemical tests to establish the identity of certain microorganisms. There have been some review papers dealing with the use of these substrates in environmental and clinical microbiology (1–3). A useful review describes the chemistry of chromogens and fluorogens in culture media (4). Some chromogenic or fluorogenic media (e.g., agar Listeria according to Ottaviani and Agosti [ALOA] and tryptone-bile-glucuronic [TBX] have therefore been taken up in the standardized ISO methods (ISO 11290-1/A1:2004 and ISO 16649:2001, respectively). This review describes some recent developments in chromogenic and fluorogenic culture media in microbiological diagnostic in particular in food and water microbiology.

GRAM-NEGATIVE BACTERIA

E. coli

E. coli is an important indicator of fecal contamination in samples from the food processing and water purification plants. The new enzymatic definition of E. coli is the possession of uidA gene coding for ß-D-glucuronidase (GUD) as an indicator for E. coli. GUD is an enzyme that catalyzes the hydrolysis of ß-D-glucopyranosiduronic acids into their corresponding aglycons and D-glucuronic acid (5, 6). GUD activity is measured by using different chromogenic and fluorogenic substances such as 4-methylumbelliferyl-ß-D-glucuronide (Fig. 1, MUG) or 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide. MUG has been incorporated into different media, including lauryl sulfate broth, lactose broth, m-Endo broth, EC broth, violet red bile agar, ECD agar, MacConkey agar and m-FC agar, and are described earlier (7). The disadvantage of incorporating MUG into solid media is that fluorescence diffuses rapidly from the colonies into the surrounding agar. The chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (BCIG) is added into TBX agar. GUD cleaves BCIG, and the released chromosphere causes distinct blue-green-colored colonies of E. coli and complies with the ISO/DIS Standard 16649 for the enumeration of E. coli in food and animal feeding stuffs.

FIGURE 1 Structure of 4-Methylumbellieryl-beta-D-glucuronide (MUG) for detection of E. coli. doi:10.1128/9781555818821.ch2.1.1.f1

Coliform

The new definition of coliforms, which is not method related, is the possession of lacZ gene coding for ß-D-galactosidase which is responsible for the cleavage of lactose into glucose and galactose. The determination of ß-D-galactosidase is accomplished by using substrates such as 6-bromo-3-indolyl-ß-galactopyranoside (Salmon-Gal, red), 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Fig. 2, blue) or fluorogenic substrate 4-methylumbelliferyl-ß-D-galactopyranoside (blue fluorescence under UV light). Attempts were made to enhance the coliform assay response by adding 1-isopropyl-ß-D-thiogalactopyronaside (IPTG) to the media (8) increasing the ß-D-galactosidase activity by improving the transfer of the substrate and/or enzyme across the outer membrane. IPTG molecule induces the lac operon but unlike the natural substrate it cannot be hydrolysed by ß-galactosidase.

FIGURE 2 Structure of X-GAL (5-bromo-4chloro-3-indolyl-beta-D-galactopyranoside) for detection of coliforms. doi:10.1128/9781555818821.ch2.1.1.f2

E. coli and Coliforms

Commercially available media have been developed which permit rapid simultaneous detection of E. coli and coliforms (Table 1) such as the Colilert system (IDEXX, Branford, CT), LMX broth, and Readycult coliforms (Merck, Germany). Other representative examples are MI agar (Becton-Dickinson, USA), CHROMagar CCA (CHROMagar, France), or Chromocult coliforms (Merck). Comparative studies have been done and are reviewed elsewhere (1). Schets et al. (47) compared Colilert with Dutch standard enumeration methods for E. coli and coliforms in water and found that Colilert gave false-negative results in samples with low numbers of E. coli or total coliforms. An evaluation of a number of presence/absence (P/A) tests for coliforms and E. coli, including LMX broth (Merck) and Colilert (IDEXX) has been published under the Department of the Environment series in the United Kingdom (48). The study concludes that there is no P/A test that is best at all locations for both coliforms and E. coli, and as there can be marked ecological differences between sources it is important that particular P/A tests are validated in each geographical area before use. The MI agar method (10) containing indoxyl-ß-D-glucuronide and 4-methylumbelliferyl-ß-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level 0.05) showed that the MI agar recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar. MI agar method is approved for detecting total coliforms and E. coli under the total coliforms rule and for enumerating total coliforms under the surface water treatment rule in the United States. Byamukama et al. (49) described the quantification of E. coli contamination with Chromocult coliform agar from different polluted sites in a tropical environment. It proved to be efficient and feasible for determining fecal pollutions in the investigated area within 24 h. Blue coloration in the broth indicates the presence of total coliforms and/or E. coli.

TABLE 1 Example of commercial chromogenic and fluorogenic culture media

E. coli O157:H7