Prescotts principles of microbiology j willey, l sherwood, c woolverton (mcgraw hill, 2009) 1

INTRODUCTION TO MICROBIOLOGYMicrobial Structure 13 MICROBIAL NUTRITION, GROWTH, AND CONTROL MICROBIAL METABOLISM MICROBIAL MOLECULAR BIOLOGY AND GENETICS THE DIVERSITY OF THE MICROBIAL

PRESCOTT’S PRINCIPLES OF MICROBIOLOGY Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas, New York, NY 10020 Copyright © 2009 by The McGraw-Hill Companies, Inc All rights reserved No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or transmission,

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Library of Congress Cataloging-in-Publication Data

ISBN 978–0–07–337523–6 — ISBN 0–07–337523–3 (hard copy : alk paper) 1 Microbiology

I Sherwood, Linda II Woolverton, Christopher J III Prescott, Lansing M Microbiology IV Title

V Title: Microbiology

QR41.2.W545 2009 616.9’041—dc22 2007040352

INTRODUCTION TO MICROBIOLOGY

Microbial Structure 13

MICROBIAL NUTRITION, GROWTH, AND CONTROL

MICROBIAL METABOLISM

MICROBIAL MOLECULAR BIOLOGY AND GENETICS

THE DIVERSITY OF THE MICROBIAL WORLD

Nonproteobacteria 420

Brief Table of Contents

ECOLOGY AND SYMBIOSIS

Microbial Ecology 593

HOST DEFENSES

MICROBIAL DISEASES AND THEIR CONTROL

APPLIED MICROBIOLOGY

Biological Molecules A-1

Glossary G-1 Credits C-1 Index I-1

About the Authors ix

Preface x

INTRODUCTION TO MICROBIOLOGY

1.1 Members of the Microbial World 2 1.2 Scope and Relevance of Microbiology 3 1.3 Discovery of Microorganisms 6

1.4 Confl ict over Spontaneous Generation 7 1.5 Golden Age of Microbiology 8

2.5 Newer Techniques in Microscopy 29

3.1 Overview of Procaryotic Cell Structure 34

Microbial Diversity & Ecology 3.1:

Monstrous Microbes 36

3.2 Procaryotic Cell Membranes 38 3.3 Procaryotic Cytoplasm 42 3.4 Bacterial Cell Walls 46 3.5 Archaeal Cell Walls 53 3.6 Components External to the Cell Wall 53 3.7 Bacterial Motility and Chemotaxis 57 3.8 Bacterial Endospores 60

4.1 Overview of Eucaryotic Cell Structure 66

4.2 Eucaryotic Membranes 67 4.3 Eucaryotic Cytoplasm 67 4.4 Organelles of the Biosynthetic-Secretory and Endocytic Pathways 70

4.5 Organelles Involved in Genetic Control of the Cell 73 4.6 Organelles Involved in Energy Conservation 75

4.7 Structures External to the Plasma Membrane 76

4.8 Comparison of Procaryotic and Eucaryotic Cells 79

5.3 Viral Multiplication 95 5.4 Types of Viral Infections 99 5.5 Cultivation and Enumeration of Viruses 103 5.6 Viroids and Virusoids 105

5.7 Prions 106

MICROBIAL NUTRITION, GROWTH, AND CONTROL

6.1 Elements of Life 110 6.2 Requirements for Carbon, Hydrogen, Oxygen, and Electrons 110

6.3 Nutritional Types of Microorganisms 111 6.4 Requirements for Nitrogen,

Phosphorus, and Sulfur 113 6.5 Growth Factors 113 6.6 Uptake of Nutrients 114 6.7 Culture Media 118

Techniques & Applications 6.1:

Microorganisms 136 7.5 Infl uences of Environmental Factors

8.4 The Use of Physical Methods in Control 157

Table of Contents

iv

Part Two

8.5 The Use of Chemical Agents in Control 160

Techniques & Applications 8.1:

Standard Microbiological Practices 162

8.6 Evaluation of Antimicrobial Agent Effectiveness 165

8.7 Biological Control of Microorganisms 167

MICROBIAL METABOLISM

9.1 Energy and Work 170 9.2 Laws of Thermodynamics 170 9.3 Free Energy and Reactions 170 9.4 ATP 171

9.5 Oxidation-Reduction Reactions 173 9.6 Electron Transport Chains 174 9.7 Enzymes 176

9.8 Ribozymes 180 9.9 Regulation of Metabolism 181 9.10 Posttranslational Regulation of Enzyme Activity 182

10.1 Chemoorganotrophic Fueling Processes 189 10.2 Aerobic Respiration 190

10.3 Breakdown of Glucose to Pyruvate 192 10.4 Tricarboxylic Acid Cycle 195

10.5 Electron Transport and Oxidative Phosphorylation 197

10.6 Anaerobic Respiration 200 10.7 Fermentation 202

10.8 Catabolism of Carbohydrates and Intracellular Reserve Polymers 206

10.9 Lipid Catabolism 207 10.10 Protein and Amino Acid Catabolism 207 10.11 Chemolithotrophy 208

Microbial Diversity & Ecology 10.1:

Acid Mine Drainage 210

10.12 Phototrophy 211

11.1 Principles Governing Biosynthesis 220 11.2 Precursor Metabolites 221

11.3 CO 2 Fixation 222 11.4 Synthesis of Sugars and Polysaccharides 224 11.5 Synthesis of Amino Acids 228 11.6 Synthesis of Purines, Pyrimidines, and Nucleotides 233

11.7 Lipid Synthesis 236

MICROBIAL MOLECULAR BIOLOGY AND GENETICS

12.1 Flow of Genetic Information 241 12.2 Nucleic Acid Structure 242

12.3 DNA Replication 245 12.4 Gene Structure 253 12.5 Transcription 255 12.6 The Genetic Code 262 12.7 Translation 263 12.8 Protein Maturation and Secretion 270

13.1 Levels of Regulation of Gene Expression 278

13.2 Regulation of Transcription Initiation 279 13.3 Regulation of Transcription Elongation 287 13.4 Regulation at the Level of Translation 289 13.5 Global Regulatory Systems 290

13.6 Regulation of Gene Expression in

Eucarya and Archaea 296

14.1 Mutations and Their Chemical Basis 301 14.2 Detection and Isolation of Mutants 306 14.3 DNA Repair 308

14.4 Creating Genetic Variability 311 14.5 Transposable Elements 312 14.6 Bacterial Plasmids 316 14.7 Bacterial Conjugation 317 14.8 Bacterial Transformation 322 14.9 Transduction 324

14.10 Mapping the Genome 326

15.1 Introduction 333 15.2 Determining DNA Sequences 333 15.3 Whole-Genome Shotgun Sequencing 335 15.4 Bioinformatics 337

15.5 Functional Genomics 337 15.6 Proteomics 344

15.7 Comparative Genomics 346 15.8 Environmental Genomics 348

16.1 Key Developments in Recombinant DNA Technology 352

16.2 Polymerase Chain Reaction 357 16.3 Gel Electrophoresis 357 16.4 Cloning Vectors and Creating Recombinant DNA 359

16.5 Construction of Genomic Libraries 363 16.6 Introducing Recombinant DNA into Host Cells 365

16.7 Expressing Foreign Genes in Host Cells 365 16.8 Microorganisms Used in Industrial

Microbiology 366

Techniques & Applications 16.1 :

Visualizing Proteins with Green

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16.12 Microbes as Products 376

Techniques & Applications 16.2:

Streptavidin-Biotin Binding and Biotechnology 378

THE DIVERSITY OF THE MICROBIAL WORLD

17.1 Microbial Evolution 382 17.2 Introduction to Microbial Classifi cation and Taxonomy 389

17.3 Taxonomic Ranks 390 17.4 Techniques for Determining Microbial Taxonomy and Phylogeny 392 17.5 Phylogenetic Trees 398 17.6 The Major Divisions of Life 399

17.7 Bergey’s Manual of Systematic Bacteriology 400

Microbial Diversity & Ecology 17.1:

“Offi cial” Nomenclature Lists—

A Letter from Bergey’s 401

18.1 Introduction to the Archaea 405 18.2 Phylum Crenarchaeota 411 18.3 Phylum Euryarchaeota 413

Microbial Diversity & Ecology 18.1 : Methanotrophic Archaea 414

19.7 Phylum Bacteroidetes 436

20.1 Class Alphaproteobacteria 440 20.2 Class Betaproteobacteria 448 20.3 Class Gammaproteobacteria 453

Microbial Diversity & Ecology 20.1 : Bacterial Bioluminescence 459

20.4 Class Deltaproteobacteria 467 20.5 Class Epsilonproteobacteria 471

21.1 Class Mollicutes (The Mycoplasmas) 475

21.2 Peptidoglycan and Endospore Structure 477

Microbial Tidbits 21.1 : Spores in Space 479

21.3 Class Clostridia 479 21.4 Class Bacilli 483

22.1 General Properties of the Actinomycetes 500

22.2 Suborder Actinomycineae 503 22.3 Suborder Micrococcineae 503 22.4 Suborder Corynebacterineae 505 22.5 Suborder Micromonosporineae 511 22.6 Suborder Propionibacterineae 511 22.7 Suborder Streptomycineae 512 22.8 Suborder Streptosporangineae 514 22.9 Suborder Frankineae 514

22.10 Order Bifi dobacteriales 514

23.1 Introduction 519 23.2 Protist Classifi cation 519

Disease 23.1 :

A Brief History of Malaria 534

23.3 Characteristics of the Fungal Divisions 540

24.1 Principles of Virus Taxonomy 555 24.2 Viruses with Double-Stranded DNA Genomes (Group I) 555

24.3 Viruses with Single-Stranded DNA Genomes (Group II) 571

24.4 Viruses with Double-Stranded RNA Genomes (Group III) 573

24.5 Viruses with Plus-Strand RNA Genomes (Group IV) 574

Microbial Diversity & Ecology 24.1 : SARS: Evolution of a Virus 579

24.6 Viruses with Minus-Strand RNA Genomes (Group V) 580

24.7 Viruses with Single-Stranded RNA Genomes (Group VI-Retroviruses) 584

24.8 Viruses with Gapped DNA Genomes (Group VII) 589

ECOLOGY AND SYMBIOSIS

Microbial Ecology 593

Microbial Diversity & Ecology 25.1 : Microbial Ecology versus Environmen- tal Microbiology 594

25.1 Biogeochemical Cycling 594 25.2 Microbial Ecology and Its Methods: An Overview 601

26.1 Marine and Freshwater Microbiology 609 26.2 Microorganisms in Terrestrial

Environments 621

Microbial Diversity & Ecology 26.1 : Mycorrhizae and the Evolution of Vascular Plants 628

vi Table of Contents

Part Five

Part Six

27 Microbial Interactions 641

27.1 Microbial Interactions 642

Microbial Diversity & Ecology 27.1 : Wolbachia pipientis: The World’s Most Infectious Microbe? 645

27.2 Human-Microbe Interactions 653 27.3 Normal Microbiota of the Human Body 654

HOST DEFENSES

28.1 Overview of Host Resistance 662 28.2 Cells, Tissues, and Organs of the Immune System 663

28.3 Phagocytosis 670 28.4 Infl ammation 673 28.5 Physical Barriers in Nonspecifi c (Innate) Resistance 675

28.6 Chemical Mediators in Nonspecifi c (Innate) Resistance 679

29.1 Overview of Specifi c (Adaptive) Immunity 690

29.2 Antigens 691 29.3 Types of Specifi c (Adaptive) Immunity 694 29.4 Recognition of Foreignness 695

29.5 T-Cell Biology 697 29.6 B-Cell Biology 701 29.7 Antibodies 704 29.8 Action of Antibodies 712

Techniques & Applications 29.1 :

Monoclonal Antibody Technology 713

29.9 Summary: The Role of Antibodies and Lymphocytes in Immune Defense 715 29.10 Acquired Immune Tolerance 716 29.11 Immune Disorders 716

MICROBIAL DISEASES AND THEIR CONTROL

30.1 Host-Parasite Relationships 727 30.2 Pathogenesis of Viral Diseases 728 30.3 Overview of Bacterial Pathogenesis 730 30.4 Toxigenicity 736

Techniques & Applications 30.1 :

Detection and Removal of Endotoxins 741

30.5 Polymicrobial Disease 742

31.1 The Development of Chemotherapy 747 31.2 General Characteristics of Antimicrobial Drugs 748

31.3 Determining the Level of Antimicrobial Activity 748

31.7 Antifungal Drugs 762 31.8 Antiviral Drugs 763 31.9 Antiprotozoan Drugs 765

32.1 Overview of the Clinical Microbiology Laboratory 769

32.2 Identifi cation of Microorganisms from Specimens 769

Techniques & Applications 32.1 : Standard Microbiological Practices 770

Historical Highlights 33.2:

“Typhoid Mary” 791

33.4 Recognition of an Epidemic 791 33.5 The Infectious Disease Cycle: Story of a Disease 793

33.6 Virulence and the Mode of Transmission 797 33.7 Emerging and Reemerging Infectious Diseases and Pathogens 798 33.8 Control of Epidemics 801

Historical Highlights 33.3:

The First Immunizations 802

33.9 Bioterrorism Preparedness 804 33.10 Nosocomial Infections 806

APPLIED MICROBIOLOGY

34.1 Microorganism Growth in Foods 810 34.2 Microbial Growth and Food Spoilage 811 34.3 Controlling Food Spoilage 814

34.4 Food-Borne Diseases 816 34.5 Detection of Food-Borne Pathogens 820 34.6 Microbiology of Fermented Foods 821

Techniques & Applications 34.1 :

Chocolate: The Sweet Side of Fermentation 822

34.7 Microorganisms as Foods and Food Amendments 829

Table of Contents vii

35 Applied Environmental Microbiology 831

35.1 Water Purifi cation and Sanitary Analysis 832

Techniques & Applications 35.1:

Waterborne Diseases, Water Supplies, and Slow Sand Filtration 833

35.2 Wastewater Treatment 836 35.3 Biodegradation and Bioremediation by Natural Communities 842

35.4 Bioaugmentation 845

Microbial Diversity & Ecology 35.2 :

A Fungus with a Voracious Appetite 846

Molecules A-1

Glossary G-1 Credits C-1 Index I-1

viii Table of Contents

Joanne M Willey is sor of Biology at Hofstra University

Profes-on LProfes-ong Island, N.Y Dr Willey received her BA in biology from the University of Pennsylvania, where her interest in microbiology began with work on cyanbacte-rial growth in eutrophic streams

She earned her PhD in cal oceanography (specializing in marine microbiology) from the Massachusetts Institute of Tech-nology–Woods Hole Oceanographic Institution Joint Program

biologi-in 1987 She then went to Harvard University, where she spent four years as a postdoctoral fellow studying the fi lamentous

soil bacterium Streptomyces coelicolor . Dr Willey continues

to actively investigate this fascinating microbe through funding provided by the National Institutes of Health and the National Science Foundation She has coauthored a number of publi-cations that focus on the complex developmental cycle of the streptomycetes She is an active member of the American So-ciety for Microbiology (ASM) and has served on the editorial

board of the journal Applied and Environmental Microbiology

since 2000 Dr Willey regularly teaches microbiology to ogy majors as well as allied health students She also teaches courses in cell biology, marine microbiology, and laboratory techniques in molecular genetics Dr Willey lives on the north shore of Long Island with her husband and two sons She is an avid runner and enjoys skiing, hiking, sailing, and reading She can be reached at biojmw@hofstra.edu

member of the Department of Microbiology at Montana State University Her interest in microbi-ology was sparked by the last course she took to complete a BS degree in psychology at Western Illinois Uni-versity She went on to complete an

MS degree in microbiology at the University of Alabama, where she

studied Pseudomonas acidovorans physiology She subsequently

earned a PhD in genetics at Michigan State University, where she

studied sporulation in Saccharomyces cerevisiae . Dr Sherwood has always had a keen interest in teaching, and her psychology training has helped her to understand current models of cognition

and learning and their implications for teaching Over the years, she has taught courses in general microbiology, genetics, biology, microbial genetics, and microbial physiology She has served as

the editor for ASM’s Focus on Microbiology Education and has

participated in and contributed to numerous ASM Conferences for Undergraduate Educators She also has worked with K-12 teach-ers to develop a kit-based unit to introduce microbiology into the elementary school curriculum and has coauthored with Barbara

Hudson a general microbiology laboratory manual, Explorations

in Microbiology: A Discovery Approach, published by

Prentice-Hall Her nonacademic interests focus primarily on her family

She also enjoys reading, hiking, gardening, and traveling She can

be reached at lsherwood@montana.edu

Christopher J

Sciences and a member of the uate faculty in Biological Sciences and the School of Biomedical Sci-ences at Kent State University in Kent, Ohio Dr Woolverton also serves as the director of the KSU Center for Public Health Prepared-ness, overseeing its BSL-3 Training Facility He earned his BS from Wilkes College, Wilkes-Barre, Pa., and a MS and a PhD in medical microbiology from West Virginia University, College of Medicine He spent two years

grad-as a postdoctoral fellow at the University of North Carolina at Chapel Hill, studying cellular immunology Dr Woolverton’s research interests are focused on the detection and control of bacterial pathogens Dr Woolverton and his colleagues have developed the fi rst liquid crystal biosensor for the immediate detection and identifi cation of microorganisms and a natural polymer system for controlled antibiotic delivery He publishes and frequently lectures on these two technologies Dr Woolver-ton has taught microbiology to science majors and allied health students, as well as graduate courses in immunology and micro-bial physiology He is an active member of ASM, serving as the

editor of ASM’s Microbiology Education He has participated in

and contributed to numerous ASM Conferences for ate Educators, serving as cochair of the 2001 conference Dr

Undergradu-Woolverton resides in Kent with his wife and three daughters

When not in the lab or classroom, he enjoys hiking, biking, kering with technology, and just spending time with his family

tin-His email address is cwoolver@kent.edu

About the Authors

ix

Prescott’s Principles of Microbiology continues in the tradition

of Prescott, Harley, and Klein’s Microbiology by covering the

broad discipline of microbiology at a depth not found in any

other textbook In using the 7th edition of PHK’s Microbiology as

the foundation for the development of Principles, we identifi ed

two overarching goals First, we sought to present material likely

to be covered in a single semester microbiology course, with the

knowledge that not all introductory microbiology courses cover

the same topics Therefore, each chapter in Prescott’s Principles

of Microbiology was revised from the 7th edition of PHK’s

Microbiology to provide a streamlined, briefer discussion of key

concepts that include only the most relevant, up-to-date

exam-ples Secondly, we strove to further extend the student-friendly

approach used in the 7th edition by enhancing readability and

adding tools designed to promote learning

OUR STRENGTHS

Connecting with Students

We have retained the relatively simple and direct writing style used

in PHK’s Microbiology, but have added style elements designed to

further engage students For example, we frequently use the fi rst

person voice to describe important concepts—especially those that

our students fi nd most diffi cult Each chapter is divided into

num-bered section headings and organized in an outline format—the

same outline format that is presented in the end-of-chapter

sum-maries Key terminology is boldfaced and clearly defi ned We have

introduced a glossary of essential terms at the beginning of each

chapter to serve as an easy reference for students, while retaining

the full glossary in the back of the book Our belief that concepts

are just as important as facts, if not more, is also refl ected in the

questions for review and refl ection that appear throughout each

chapter These questions are of two types: those that quiz student

retention of key facts and vocabulary and those designed to foster

critical thinking

Instructive Artwork

To truly engage students, a textbook must do more than offer

words and images that just adequately describe the topic at hand

We view the artwork of a text as a critical tool in enticing students

to read the text Principles features the art program introduced

in the 7th edition of PHK’s Microbiology The three-dimensional

renderings help students appreciate the beauty and elegance of

the cell, while at the same time make the material more

com-prehendible Of course we also believe that fi gures should be

content-rich, not just pretty to look at Therefore, the art program

also includes pedagogical features such as concept maps (e.g.,

see fi gures 9.1 and 13.1) and annotation of key pathways and

processes (e.g., see fi gures 10.8 and 12.12)

Unique Organization Around Key Themes

With the advent of genomics, proteomics, metabolomics and the increased reach of cell biology, the divisions among microbiology subdisciplines have become blurred This is refl ected in the emer-gence of fi elds like disease ecology and metagenomics In addition, today’s microbiologist must be acquainted with all members of the microbial world: viruses, bacteria, archaea, protists, and fungi It follows that students new to microbiology are asked to assimilate vocabulary, facts, and most importantly, concepts, from a seemingly vast array of subjects The challenge to the professor of microbiol-ogy is to effectively communicate essential concepts while convey-ing the ingenuity of microbes and excitement of this dynamic fi eld

Microbial Evolution and EcologyBecause microbial evolution and ecology are no longer subdis-ciplines to be ignored by those interested in microbial genetics,

physiology, or pathogenesis, Principles strives to integrate these

themes throughout the text We begin in chapter 1 with a sion of the universal tree of life and whenever possible, discuss diverse microbial species so that students can begin to appreciate

discus-the tremendous variation in discus-the microbial world In addition, ciples uses the topics of intercellular communication (chapters 6

Prin-and 13), biofi lms (throughout the text, but specifi cally in chapters

6, 13, and 29), microbial evolution (chapter 17), and polymicrobial diseases (chapter 33) to emphasize that evolution must be linked

to genetics, physiology to diversity, and ecology to pathogenesis

Microbial Pathogenicity and Diversity

Unique to Principles is the inclusion of microbial pathogens into

the diversity chapters (chapters 19–24) Thus when students read about the metabolic and genetic diversity of each bacterial, pro-tist, and viral taxon, they are also presented with the important pathogens In this way, the physiological adaptations that make a given organism successful can be immediately related to its role

as a pathogen and pathogens can be readily compared to genetically related nonpathogenic microbes

In addition, Principles introduces viruses and other acellular

agents in chapter 5, following the chapters of Procaryotic and Eucaryotic Cell Structure and Function (chapters 3 and 4, respec-tively) By placing a similarly themed chapter on viruses here, professors can introduce all divisions of the microbial world to their students early in the term For those professors who include more in-depth coverage of viruses, chapter 24 explores the mo-lecular genetics of bacteriophages and other viruses as well as the

pathogenicity of important animal and plant viruses As in PHK’s Microbiology, we use the classifi cation schemes set forth in the second edition of Bergey’s Manual of Systematic Bacteriology,

the Baltimore System of virus classifi cation (chapters 5 and 24), and the International Society of Protistologists’ new classifi ca-tion scheme for eucaryotes (chapter 23)

Preface

x

a-head # A-head Title Goes Here xi

Visual Tour

xi

INSTRUCTOR RESOURCES

ARIS

McGraw-Hill’s ARIS (Assessment, Review, and Instruction

Sys-tem) for Prescott’s Principles of Microbiology is a complete, online

tutorial, electronic homework, and course management system, designed for greater ease of use than any other system available For students, ARIS contains self-study tools such as animations, interac-tive quizzes, and more This program enables students to complete their homework online, as assigned by their instructors ARIS pro-vides all instructor resources online, as well provides the ability to create or edit questions from the question bank, import your own content, and automatically grade and report easy-to-assign home-work, quizzing, and testing

Go to www.aris.mhhe.com to learn more

McGraw-Hill’s ARIS—Assessment, Review, and Instruction

System—for Prescott’s Principles of Microbiology provides

helpful online study materials and resources that support each chapter in the book Features include:

STUDENT RESOURCES

Laboratory Exercises in Microbiology

The seventh edition of Laboratory Exercises in Microbiology by John P Harley has been

prepared to accompany the text Like the text, the laboratory manual provides a balanced introduction in each area of microbiology The class-tested exercises are modular and short so that instructors can easily choose those exercises that fi t their course

enhanced tests and quizzes, compelling course websites, or tive printed support materials

attrac-Access to your book, access to all books! This ever-growing

resource gives instructors the power to utilize assets specifi c

to their adopted textbook as well as content from other McGraw-Hill books in the library Presentation Center’s dynamic search engine allows you to explore by discipline, course, textbook chapter, asset type, or keyword Simply browse, select, and download the fi les you need to build engag-ing course materials All assets are copyrighted by McGraw-Hill Higher Education but can be used by instructors for classroom purposes

Instructor’s Manual

The Instructor’s Manual is available in both Word and PDF formats and contains chapter overviews, objectives, and answer guide-lines for Critical Thinking Questions

xii Chapter # Chapter Title

xii Visual Tour

bank allows the user to quickly create customized exams This

user-friendly program allows instructors to search for questions

by topic, format, or diffi culty level; edit existing questions or add

new ones; and scramble questions and answer keys for multiple

versions of the same test

Transparencies

A set of transparency masters can be customized for your

course Please contact your McGraw-Hill sales representative for

details

Electronic Books

If you or your students are ready for an alternative version of the

traditional textbook, McGraw-Hill and VitalSource have

part-nered to bring you innovative and inexpensive electronic

text-books By purchasing E-books from McGraw-Hill & VitalSource, students can save as much as 50% on selected titles delivered

on the most advanced E-book platform available, VitalSource Bookshelf

E-books from McGraw-Hill & VitalSource are smart, interactive, searchable, and portable VitalSource Bookshelf comes with a powerful suite of built-in tools that allow detailed searching, highlighting, note taking, and student-to-student or instructor-to-student note sharing E-books from McGraw-Hill

& VitalSource will help students study smarter and quickly

fi nd the information they need And they will save money

Contact your McGraw-Hill sales representative to discuss E-book packaging options

1

Dans les champs de l’observation, le hasard ne favorise que les esprits préparés

(In the field of observation, chance favors only prepared minds.)

—Louis Pasteur

Louis Pasteur, one of the greatest scientists of the nineteenth century,

maintained that “Science knows no country, because knowledge

be-longs to humanity, and is a torch which illuminates the world.”

Chapter Glossary

The History and Scope

of Microbiology

The importance of microorganisms cannot be overemphasized In

50% of the biological carbon and 90% of the biological nitrogen on

planet Furthermore, they are found everywhere: from geothermal

contributors to the functioning of the biosphere, being

indispens-able for the cycling of the elements essential for life They also are

a source of nutrients at the base of all ecological food webs Most important, certain microorganisms carry out photosynthesis, rivaling into the atmosphere Those microbes that inhabit humans are also and K In addition, society in general benefi ts from microorganisms

Indeed, modern biotechnology rests upon a microbiological tion, as microbes are necessary for the production of bread, cheese,

founda-Archaea The domain of life that contains procaryotic cells with

cell walls that lack peptidoglycan; they have unique lipids in their membranes and archaeal rRNA (among many differ- ences) 00

Bacteria The domain of life that contains procaryotic cells with

cell walls that contain the structural molecule peptidoglycan;

they have bacterial rRNA 00

Eucarya The domain of life that features organisms made of

cells that have a membrane-delimited nucleus and differ in fungi, plants, and animals 00

fungi A diverse group of microorganisms that range from

unicellular forms (yeasts) to multicellular molds and mushrooms 00

Koch’s postulates A set of rules for proving that a specifi c

microorganism causes a particular disease 00

microbiology The study of organisms that are usually too

small to be seen with the naked eye; special techniques are required to isolate and grow them 00

microorganism An organism that is too small to be seen

clearly with the naked eye and lacks highly differentiated cells and distinct tissues 00

prions Infectious agents that cause spongiform

encephalopa-thies such as scrapie in sheep; they are composed only of protein 00

procaryotic cells Cells that lack a true, membrane-enclosed

nucleus; Bacteria and Archaea are procaryotic and have their

genetic material located in a nucleoid 00

protists Mostly unicellular eucaryotic organisms that lack

cel-lular differentiation into tissues; cell differentiation is limited to cells involved in sexual reproduction, alternate vegetative mor- phology, or resting states such as cysts; includes organisms often referred to as algae and protozoa 00

spontaneous generation An early belief, now discredited,

that living organisms could develop from nonliving matter 00

viroids Infectious agents composed only of single-stranded,

circular RNA; they cause numerous plant diseases 00

viruses Infectious agents having a simple acellular

organiza-tion with a protein coat and a nucleic acid genome, lacking host cells 00

virusoids Infectious agents composed only of single-stranded

RNA; they are unable to replicate without the aid of specifi c viruses that coinfect the host cell 00

1

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Anoxygenic phototrophs have photosynthetic pigments called

bacteriochlorophylls ( fi gure 10.27 ) In some bacteria, these are

located in membranous vesicles called chlorosomes The tion maxima of bacteriochlorophylls (Bchl) are at longer

absorp-wavelengths than those of chlorophylls Bacteriochlorophylls a and b have maxima in ether at 775 and 790 nm, respectively In vivo maxima are about 830 to 890 nm (Bchl a ) and 1,020 to 1,040

nm (Bchl b ) This shift of absorption maxima into the infrared

region better adapts these bacteria to their ecological niches

>> Photosynthetic bacteria ( section 19.3 )

Many differences found in anoxygenic phototrophs are because they have a single photosystem Because of this, they produce O 2 from H 2 O Indeed, almost all anoxygenic photo- trophs are strict anaerobes A tentative scheme for the photosynthetic ETC of a purple nonsulfur bacterium is given in

fi gure 10.31 When the reaction-center bacteriochlorophyll P870 is excited, it donates an electron to bacteriopheophytin

P870 while generating suffi cient PMF to drive ATP synthesis by lack two photosystems, the purple bacteria have a photosynthetic apparatus similar to photosystem II of oxygenic phototrophs, whereas the green sulfur bacteria have a system similar to photosystem I >> Class Alphaproteobacteria: Purple honsulfur bacteria ( section 20.1 )

Anoxygenic photoautotrophs face a further problem because they also require reducing power (NAD[P]H or reduced ferre- doxin) for CO 2 fi xation and other biosynthetic processes They are able to generate reducing power in at least three ways, depending on the bacterium Some have hydrogenases that are used to produce NAD(P)H directly from the oxidation of hydro- gen gas This is possible because hydrogen gas has a more negative reduction potential than NAD ⫹ ( see table 9.1 ) Others,

such as the photosynthetic purple bacteria, use reverse electron

fl ow to generate NAD(P)H ( fi gure 10.31 ) In this mechanism,

to NAD(P) ⫹ using PMF Electrons from electron donors such as

Thylakoid Stroma

ATP synthase

Pyridine nucleotide reductase

2H +

Figure 10.30 The Mechanism of Photosynthesis. An illustration of the chloroplast thylakoid membrane showing photosynthetic ETC function and noncyclic photophosphorylation The chain is composed of three complexes: PS I, the cytochrome bf complex, and PS II Two diffusible electron carriers

connect the three complexes Plastoquinone (PQ) connects PS I with the cytochrome bf complex, and plastocyanin (PC) connects the cytochrome bf complex

with PS II The light-driven electron fl ow pumps protons across the thylakoid membrane and generates an electrochemical gradient, which can then be used

to make ATP Water is the source of electrons and the oxygen-evolving complex (OEC) produces oxygen

10.12 Phototrophy 215

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TOOLS FOR LEARNING

Chapter Glossary

Each chapter begins with a glossary—a list of key terms discussed

in the chapter Each term is succinctly defined

Cross-Referenced Notes

In-text references with icons refer students to other parts of the book to review

a-head # A-head Title Visual Tour xiii Goes Here xiii

ACKNOWLEDGMENTS

We would like to thank the Board of Advisors, who provided constructive reviews of every chapter, including the line art and photos in the book Their specialized knowledge helped assimi-late more reliable sources of informaton, and fi nd more effective ways of expressing an idea for the student reader

Board of Advisors

Morad Abou-Sabe, Rutgers University Shivanthi Anandan, Drexel University Penny Antley, University of Louisiana–Lafayette Phil Cunningham, Wayne State University Bernard Frye, University of Texas at Arlington Mike Henson, Clemson University

Mike Hyman, North Carolina State University–Raleigh Michael Ibba, The Ohio State University

Jeffrey Leblond, Middle Tennessee State University

S N Rajagopal, University of Wisconsin–Lacrosse William Safranek, University of Central Florida Lisa Stein, University of California–Riverside Herman Witmer, University of Illinois at Chicago

We owe our collective thanks to Lisa Brufl odt, Jim Connely, Tami Petsche, Mary Powers, Mary Jane Lampe and Sandy Ludovissy

We would also like to thank our design editor John Joran and photo editor Mary Reeg

This text is dedicated to our families for their patience and to our students for teaching us how to teach better

Review and Refl ection Questions Within Narrative

Review questions throughout each chapter assist students in tering section concepts before moving on to other topics

mas-376 Chapter 16 Biotechnology and Industrial Microbiology

1 What is the Ti plasmid and how is modifi ed for the genetic modifi cation of plants?

2 How is the Bt toxin produced and why is it so widely accepted?

3 Can you think of other traits that might be useful, either

to the farmer or the consumer, that could be introduced into plants?

16.12 MICROBES AS PRODUCTS

So far, we have discussed the use of microbial products to meet defi ned goals However, microbial cells can be marketed as valu- able products Perhaps the most common example is the inoculation

of legume seeds with rhizobia to ensure effi cient nodulation and several other microbes and microbial structures that are of indus- trial or agricultural relevance

Diatoms have aroused the interest of nanotechnologists

These photosynthetic protists produce intricate silica shells that differ according to species ( fi gure 16.22 ) Nanotechnologists are interested in diatoms because they create precise structures at the micrometer scale Three-dimensional structures in nanotechnol- ogy are currently built plane by plane, and meticulous care must Diatoms, on the other hand, build directly in three dimensions and do so while growing exponentially There have been a num- ber of ideas and approaches to harness these microbial “factories,”

but one technique is especially fascinating Diatoms shells are hours Amazingly, this results in an atom-for-atom substitution of

(a)

Toxins binding to phospholipids and insertion into membrane

NH 2 COOH

Plasma membrane

Osmotic imbalance and cell lysis

Gut eptihelial plasma membrane

H 2 O, cations

H 2 O, cations

Inside cell

Inside cell Toxin protein ion channel Inside cell

Outside cell Outside cell

Outside cell

Aggregation and pore formation

Efflux of ATP

Parasporal crystal

Alkaline gut contents

250 kDa subunit protoxin

Figure 16.21 The Mode of Action of the Bacillus

thuringiensis Toxin. (a) Release of the protoxin from the parasporal body and modifi cation by proteases in the hindgut

(b) Insertion of the 68 kDa active toxin molecules into the brane (c) Aggregation and pore formation, showing a cross section of the pore (d) Final hexagonal pore, which causes an in-

mem-fl ux of water and cations as well as a loss of ATP, resulting in cell imbalance and lysis.

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1 Examine the carbon cycle shown in fi gure 25.1 What do you think are some major biogenic sources of CO 2 emission into the atmosphere? What are the major biogenic sinks of atmospheric

of carbon for methanogenesis in a waterlogged peat?

Critical Thinking Questions

b Biogeochemical cycling involves oxidation and reduction processes, and changes in the concentrations of gaseous cycle components, such as carbon, nitrogen, phosphorus,

and sulfur, can result from microbial activity (figures

25.2 Microbial Ecology and Its Methods:

An Overview

a A variety of staining techniques are used to observe microbes in natural environments Fluorescent in situ hybridization (FISH) labels specifi c microbes that possess a specifi c nucleotide se-

quence, u sually a region of the SSU rRNA gene ( fi gure 25.9 )

b Although most microbes cannot be grown in pure culture, richment techniques are invaluable in isolating microorganisms

c The analysis of rRNA from natural samples can be plished after amplifi cation by PCR (to determine nucleotide

accom-sequence) or by DNA fi ngerprinting ( fi gure 25.13 )

d DNA reassociation is also used to evaluate the size and plexity of microbial communities This technique considers the community as a collection of genomes

e Microelectrodes can be used to determine physical and logical parameters such as pH, O 2 , and H 2 S concentrations in

xiv Chapter # Chapter Title

xiv Visual Tour

Special Interest Essays

Interesting essays on relevant topics are included in most chapters Readings are

orga-nized into these topics: Historical Highlights, Techniques & Applications, Microbial

Diversity & Ecology, Disease, and Microbial Tidbits

The basidiomycete Phanerochaete chrysosporium (the scientifi c name

capabilities This organism is termed a “white rot fungus” because of its

ability to degrade lignin, a randomly linked, phenylpropene-based

poly-meric component of wood The cellulosic portion of wood is attacked to

wood This organism also degrades a truly amazing range of xenobiotic

compounds (nonbiological foreign chemicals) using both intracellular

and extracellular enzymes.

As examples, the fungus degrades benzene, toluene, ethylbenzene,

and xylenes (the so-called BTEX compounds), chlorinated compounds

such as 2,4,5-trichloroethylene (TCE), and trichlorophenols (fi gure

are used as pesticides In addition, other chlorinated benzenes can be

Hydramethylnon is degraded.

How does this microorganism carry out such feats? Apparently

most xenobiotic degradation occurs after active growth, during

second-ary metabolic lignin degradation Degradation of some compounds

35.2 A Fungus with a Voracious Appetite

Microbial Diversity & Ecology

involves important extracellular enzymes including lignin peroxi- oxidase, and glyoxal oxidase A critical enzyme is pyranose oxi- dase, which releases H 2 O 2 for use by the manganese-dependent peroxidase enzyme The H 2 O 2 also is a precursor of the highly reactive hydroxyl radical, which participates in wood degradation Apparently the pyranose oxidase enzyme is located in the interperiplasmic space of

or be released and penetrate into the wood substrate It appears that the nonspecifi c enzymatic system that releases these oxidizing products degrades many cyclic, aromatic, and chlorinated compounds related to lignins.

We can expect to continue hearing of many new advances regarding this organism Potentially valuable applications being studied include growth in bioreactors, where intracellular and extracellular enzymes can

be maintained in the bioreactor while liquid wastes fl ow past the bilized fungi.

immo-organism physical protection, as well as possibly supplying

nutrients This makes it possible for the microorganism to

sur-vive in spite of the intense competitive pressures that exist in the

natural environment, including pressure from protozoan

preda-tors Microhabitats may be either living or inert Specialized

living microhabitats include the surface of a seed, a root, or a

leaf Here, higher nutrient fl uxes and rates of initial colonization

by the added microorganisms protect against the fi erce

competi-tive conditions in the natural environment For example, to

ensure that the nitrogen-fi xing microbe Rhizobium is in close

using an oil-organism mixture or the bacteria are placed in a

will penetrate.<< Microorganisms in terrestrial environments: The

Rhizobia (section 26.2)

Recently it has been found that microorganisms can be added

to natural communities together with protective inert

microhabi-tats As an example, if microbes are added to a soil with microporous glass, the survival of added microorganisms can be markedly enhanced Other microbes have been observed to cre- ate their own microhabitats Microorganisms in the water column

to create their own “clay hutches” by binding clays to their outer surfaces with exopolysaccharides Thus the application of principles of microbial ecology can facilitate the successful man- agement of microbial communities in nature

1 What factors might limit the ability of microorganisms, after addition to a soil or water, to persist and carry out desired functions?

2 What types of microhabitats can be used with organisms when they are added to a complex natural environment?

micro-wiL75233_ch35_831-847.indd Page 846 10/9/07 6:13:44 AM elhi /Volumes/ve401/MHIY034/mhwiL1%0/wiL1ch35

Historical Highlights

Much of what we know today about the epidemiology of cholera is Snow between 1849 and 1854 During this period, a series of cholera source of the disease Some years earlier when he was still a medical among coal miners His observations convinced him that the disease air or casual direct contact

Thus when the outbreak of 1849 occurred, Snow believed that cholera was spread among the poor in the same way as among the shared food, was the source of the cholera infection among the wealthier residents Snow examined offi cial death records and dis- covered that most of the victims in the Broad Street area had lived drinking from it He concluded that cholera was spread by drinking sewage containing the disease agent When the pump handle was removed, the number of cholera cases dropped dramatically

In 1854 another cholera outbreak struck London Part of the city’s water supply came from two different suppliers: the Southwark and cholera patients and found that most of them purchased their drinking that this company obtained its water from the Thames River below the Lambeth Company took its water from the Thames before the river

in households supplied with Lambeth Company water Water nated by sewage was transmitting the disease Finally, Snow concluded nearly recognized that cholera was caused by a microorganism, though

contami-Robert Koch did not discover the causative bacterium ( Vibrio

chol-erae ) until 1883

To commemorate these achievements, the John Snow Pub now stands at the site of the old Broad Street pump Those who complete the Epidemiologic Intelligence Program at the Centers for Disease Control and Prevention receive an emblem bearing a replica of a bar- rel of Whatney’s Ale—the brew dispensed at the John Snow Pub

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Disease

31.1 Antibiotic Misuse and Drug Resistance

The sale of antimicrobial drugs is big business In the United States,

millions of pounds of antibiotics valued at billions of dollars are

pro-duced annually As much as 70% of these antibiotics are added to

livestock feed

Because of the massive quantities of antibiotics being prepared

and used, an increasing number of diseases are resisting treatment

due to the spread of drug resistance A good example is Neisseria

gonorrhoeae, the causative agent of gonorrhea Gonorrhea was fi rst

treated successfully with sulfonamides in 1936, but by 1942 most

16 years, a resistant strain emerged in Asia A

penicillin-ase-producing gonococcus reached the United States in 1976 and is

still spreading in this country Thus penicillin is no longer used to

treat gonorrhea

In late 1968 an epidemic of dysentery caused by Shigella broke

out in Guatemala and affected at least 112,000 persons; 12,500

deaths resulted The strains responsible for this devastation carried

an R plasmid conferring resistance to chloramphenicol, tetracycline,

streptomycin, and sulfonamide In 1972 a typhoid epidemic swept

was due to a Salmonella strain with the same multiple-drug-resistance

pattern seen in the previous Shigella outbreak

Haemophilus infl uenzae type b is responsible for many cases

of childhood pneumonia and middle ear infections, as well as spiratory infections and meningitis It is now becoming increasingly the worldwide rate of penicillin-nonsusceptible (i.e., resistant)

Streptococcus pneumoniae (PNSP) continues to increase There is a

direct correlation between the daily use of antibiotics (expressed as defi ned daily dose [DDD] per day) and the percent of PNSP isolates cultured ( box fi gure ) This dramatic correlation is alarming More alarming is the continued indiscriminant use of antibiotics in light of these data

In 1946 almost all strains of Staphylococcus were penicillin

sensitive Today most hospital strains are resistant to penicillin G,

only can be treated with vancomycin Strains of Enterococcus have

become resistant to most antibiotics, including vancomycin, and a the United States and Japan

It is clear from these and other examples (e.g., multiresistant

Mycobacterium tuberculosis ) that drug resistance is an extremely

serious public health problem Much of the diffi culty arises from

of Bacillus subtilis spores Their data suggest that spores within an

interstellar molecular cloud might be able to survive between 4.5 to

45 million years Molecular clouds move through space at speeds suffi cient to transport spores between solar systems in this length of they are consistent with the possibility that bacteria might be able to travel between planets capable of supporting life.

21.1 Spores in Space

During the nineteenth-century argument over the question of the lution of life, the panspermia hypothesis became popular According but arrived as viable bacterial spores that escaped from another planet More recently the British astronomer Fred Hoyle has revived the hypothesis based on his study of the absorption of radiation by interstellar dust Hoyle maintains that dust grains were initially via- ble bacterial cells that have been degraded and that the beginning of life on Earth was due to the arrival of bacterial spores that had sur- vived their trip through space.

evo-Techniques and Applications

enzyme (box fi gure) Active clotting enzyme then catalyzes the cleavage of procoagulogen into polypeptide subunits (coagulo- gen) The subunits join by disulfi de bonds to form a gel-clot

Spectrophotometry is then used to measure the protein precipitated must be standardized against U.S Food and Drug Administration reported in endotoxin units per milliliter and reference made to the particular reference standards used.

Removal of endotoxins presents more of a problem than their detection Those present on glassware or medical devices can be inactivated if the equipment is heated at 250°C for 30 minutes

Soluble endotoxins range in size from 20 kDa to large aggregates with diameters up to 0.1 ␮m Thus they cannot be removed by con- ventional fi ltration systems Manufacturers have developed special

fi ltration systems and fi ltration cartridges that retain these endotoxins and help alleviate contamination problems.

30.1 Detection and Removal of Endotoxins

Bacterial endotoxins plagued the pharmaceutical industry and cal device producers for years For example, administration of drugs contaminated with endotoxins resulted in complications—even death—to patients In addition, endotoxins can be problematic for individuals and fi rms working with cell cultures and genetic engi- neering The result has been the development of sensitive tests and must be very sensitive to trace amounts of endotoxins Most fi rms have set a limit of 0.25 endotoxin units (E.U.), 0.025 ng/ml, or less

medi-as a relemedi-ase standard for their drugs, media, or products.

One of the most accurate tests for endotoxins is the in vitro

Limulus amoebocyte lysate (LAL) assay The assay is based on the

observation that when an endotoxin contacts the clot protein from forms The assay kits contain calcium, proclotting enzyme, and endotoxin (lipopolysaccharide) and calcium to form active clotting wiL75233_ch30_726-745.indd Page 741 10/9/07 6:22:55 AM elhi /Volumes/ve401/MHIY034/mhwiL1%0/wiL1ch30

PRESCOTT’S PRINCIPLES OF MICROBIOLOGY Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas, New York, NY 10020 Copyright © 2009 by The McGraw-Hill Companies, Inc All rights reserved No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or transmission,

or broadcast for distance learning.

Some ancillaries, including electronic and print components, may not be available to customers outside the United States.

This book is printed on acid-free paper.

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www.mhhe.com

ISBN 978–0–07–128367–0 MHID 0–07–128367–6 The credits section for this book begins on page C-1 and is considered an extension of the copyright page.

Dans les champs de l’observation, le hasard ne favorise que les esprits préparés

(In the field of observation, chance favors only prepared minds.)

—Louis Pasteur

Louis Pasteur, one of the greatest scientists of the nineteenth century, maintained that “Science knows no country, because knowledge be- longs to humanity, and is a torch which illuminates the world.”

a source of nutrients at the base of all ecological food webs Most important, certain microorganisms carry out photosynthesis, rivaling plants in their role of capturing carbon dioxide and releasing oxygen into the atmosphere Those microbes that inhabit humans are also important, helping the body digest food and producing vitamins B and K In addition, society in general benefi ts from microorganisms

Indeed, modern biotechnology rests upon a microbiological tion, as microbes are necessary for the production of bread, cheese,

founda-Archaea The domain of life that contains procaryotic cells with

cell walls that lack peptidoglycan; they have unique lipids

in their membranes and archaeal rRNA (among many differences).

Bacteria The domain of life that contains procaryotic cells with

cell walls that contain the structural molecule peptidoglycan;

they have bacterial rRNA.

Eucarya The domain of life that features organisms made of

cells that have a membrane-delimited nucleus and differ in many other ways from procaryotic cells; includes protists, fungi, plants, and animals.

fungi A diverse group of microorganisms that range from

unicellular forms (yeasts) to multicellular molds and mushrooms.

Koch’s postulates A set of rules for proving that a specifi c

microorganism causes a particular disease.

microbiology The study of organisms that are usually too

small to be seen with the naked eye; special techniques are required to isolate and grow them.

microorganism An organism that is too small to be seen

clearly with the naked eye and lacks highly differentiated cells and distinct tissues.

prions Infectious agents that cause spongiform

encephalopa-thies such as scrapie in sheep; they are composed only of protein.

procaryotic cells Cells that lack a true, membrane-enclosed

nucleus; Bacteria and Archaea are procaryotic and have their

genetic material located in a nucleoid.

protists Mostly unicellular eucaryotic organisms that lack

cel-lular differentiation into tissues; cell differentiation is limited to cells involved in sexual reproduction, alternate vegetative mor- phology, or resting states such as cysts; includes organisms often referred to as algae and protozoa.

spontaneous generation An early belief, now discredited,

that living organisms could develop from nonliving matter.

viroids Infectious agents composed only of single-stranded,

circular RNA; they cause numerous plant diseases.

viruses Infectious agents having a simple acellular

organiza-tion with a protein coat and a nucleic acid genome, lacking independent metabolism, and reproducing only within living host cells.

virusoids Infectious agents composed only of single-stranded

RNA; they are unable to replicate without the aid of specifi c viruses that coinfect the host cell.

1

2 Chapter 1 The History and Scope of Microbiology

beer, antibiotics, vaccines, vitamins, enzymes, and many other

prod-ucts Their ability to produce biofuels such as ethanol is also being

intensively explored These alternative fuels are both renewable and

can help decrease pollution associated with burning fossil fuels

Although most microorganisms play benefi cial or benign roles,

some harm humans and have disrupted society over the

millen-nia Microbial diseases undoubtedly played a major role in historical

events such as the decline of the Roman Empire and the conquest

of the New World In 1347, plague (Black Death), an

arthropod-borne disease, struck Europe with brutal force, killing one-third of the

population (about 25 million people) within four years Over the next

80 years, the disease struck repeatedly, eventually wiping out 75%

of the European population The plague’s effect was so great that

some historians believe it changed European culture and prepared

the way for the Renaissance Today the struggle by microbiologists

and others against killers such as AIDS and malaria continues

In this chapter, we introduce the microbial world to provide a

gen-eral idea of the organisms and agents that microbiologists study We

next discuss the scope and relevance of modern microbiology Finally,

we describe the historical development of the science of microbiology

and its relationship to medicine and other areas of biology

MICROBIAL WORLD Microbiology often has been defi ned as the study of organisms

and agents too small to be seen clearly by the unaided eye—that

is, the study of microorganisms Because objects less than about

1 millimeter in diameter cannot be seen clearly and must be

exam-ined with a microscope, microbiology is concerned primarily with

organisms and agents this small and smaller However, some

microorganisms, particularly some eucaryotic microbes, are visible

without microscopes For example, bread molds and fi lamentous

algae are studied by microbiologists yet are visible to the naked

eye, as are the two bacteria Thiomargarita and Epulopiscium

>> Microbial Diversity & Ecology 3.1: Monstrous Microbes

The diffi culty in setting the boundaries of microbiology has led

to the suggestion of other criteria for defi ning the fi eld For instance,

an important characteristic of microorganisms, even those that are

large and multicellular, is that they are relatively simple in their

construction, lacking highly differentiated cells and distinct

tis-sues Another suggestion, made by Roger Stanier, is that the fi eld

also be defi ned in terms of its techniques Microbiologists usually

fi rst isolate a specifi c microorganism from a population and then

culture it Thus microbiology employs techniques—such as

sterilization and the use of culture media—that are necessary for

successful isolation and growth of microorganisms

Microorganisms are diverse, and their classifi cation has always

been a challenge for microbial taxonomists Their early

descrip-tions as either plants or animals were too simple For instance,

some microbes are motile like animals but also have cell walls and

are photosynthetic like plants Such microbes cannot be placed easily into one kingdom or another Another important factor in classifying microorganisms is that some are composed of procary-

otic cells and others of eucaryotic cells Procaryotic cells (Greek

pro, before, and karyon, nut or kernel; organisms with a primordial

nucleus) have a much simpler morphology than eucaryotic cells

and lack a true membrane-delimited nucleus In contrast,

membrane-enclosed nucleus; they are more complex cally and are usually larger than procaryotes These observations eventually led to the development of a classifi cation scheme that

morphologi-divided organisms into fi ve kingdoms: the Monera, Protista, Fungi, Animalia, and Plantae Microorganisms (except for viruses and

other acellular infectious agents, which have their own classifi tion system) were placed in the fi rst three kingdoms

In the last few decades, great progress has been made in three areas that profoundly affect microbial classifi cation First, much has been learned about the detailed structure of microbial cells from the use of electron microscopy Second, microbiologists have deter-mined the biochemical and physiological characteristics of many different microorganisms Third, the sequences of nucleic acids and proteins from a wide variety of organisms have been compared

The comparison of ribosomal RNA (rRNA), begun by Carl Woese

in the 1970s, was instrumental in demonstrating that there are two

very different groups of procaryotic organisms: Bacteria and Archaea, which had been classifi ed together as Monera in the fi ve-

kingdom system Later studies based on rRNA comparisons showed

that Protista is not a cohesive taxonomic unit and that it should be

divided into three or more kingdoms These studies and others have led many taxonomists to conclude that the fi ve-kingdom system is too simple A number of alternatives have been suggested, but cur-rently most microbiologists believe that organisms should be

divided among three domains: Bacteria (the true bacteria or eubacteria), Archaea, 1

and Eucarya (all eucaryotic organisms)

( fi gure 1.1 ) We use this system throughout the text, and it is cussed in detail in chapter 17 However, a brief description of the three domains and of the microorganisms placed in them follows

dis-Bacteria 2 are procaryotes that are usually single-celled isms Most have cell walls that contain the structural molecule peptidoglycan They are abundant in soil, water, and air, and are major inhabitants of our skin, mouth, and intestines Some bacte-ria live in environments that have extreme temperatures, pH, or salinity Although some bacteria cause disease, many more play benefi cial roles such as cycling elements in the biosphere, break-ing down dead plant and animal material, and producing vitamins

organ-Cyanobacteria (once called blue-green algae) produce signifi cant amounts of oxygen through the process of photosynthesis

Archaea are procaryotes that are distinguished from Bacteria

by many features, most notably their unique ribosomal RNA sequences They lack peptidoglycan in their cell walls and have unique membrane lipids Some have unusual metabolic

this text, we use only the name Archaea.

2

In this text, the term bacteria (s., bacterium) is used to refer to procaryotes that belong to domain Bacteria, and the term archaea (s., archaeon) is used to refer to procaryotes that belong to

characteristics, such as the methanogens, which generate ane gas Many archaea are found in extreme environments, including those with high temperatures (thermophiles) and high concentrations of salt (extreme halophiles) Pathogenic archaea have not yet been identifi ed

Domain Eucarya includes microorganisms classifi ed as

pro-tists or fungi Animals and plants are also placed in this domain

Photosynthetic protists, together with the cyanobacteria, produce about 75% of the planet’s oxygen These phytoplankton are the

foundation of aquatic food chains Protozoa are unicellular,

animal-like protists that are usually motile Many free-living protozoa function as the principal hunters and grazers of the microbial world

They obtain nutrients by ingesting organic matter and other microbes They can be found in many different environments, and some are normal inhabitants of the intestinal tracts of animals, where they aid in digestion of complex materials such as cellulose

A few cause disease in humans and other animals Slime molds are

protists that are like protozoa in one stage of their life cycle but like fungi in another In the protozoan phase, they hunt for and engulf food particles, consuming decaying vegetation and other microbes

Water molds are protists that grow on the surface of freshwater

and moist soil They feed on decaying vegetation such as logs and mulch Some water molds have produced devastating plant infec-

tions, including the Great Potato Famine of 1846–1847 in Ireland

Fungi are a diverse group of microorganisms that range from

unicellular forms (yeasts) to molds and mushrooms Molds and mushrooms are multicellular fungi that form thin, threadlike struc-tures called hyphae They absorb nutrients from their environment, including the organic molecules that they use as a source of carbon and energy Because of their metabolic capabilities, many fungi play benefi cial roles, including making bread rise, producing anti-biotics, and decomposing dead organisms Some fungi associate with plant roots to form mycorrhizae Mycorrhizal fungi transfer nutrients to the roots, improving the growth of the plants, especially

in poor soils Other fungi cause plant diseases (e.g., rusts, powdery mildews, and smuts) and diseases in humans and other animals

The microbial world also includes numerous acellular

infec-tious agents Viruses are acellular entities that must invade a host

cell to replicate The simplest viruses are composed only of teins and a nucleic acid, and can be extremely small (the smallest

pro-is 10,000 times smaller than a typical bacterium) However, their small size belies their power—they cause many animal and plant diseases and have caused epidemics that have shaped human his-tory The diseases they cause include smallpox, rabies, infl uenza, AIDS, the common cold, and some cancers Viroids and virusoids are infectious agents composed only of ribonucleic

acid (RNA) Viroids cause numerous plant diseases, whereas virusoids cause some important animal diseases such as hepatitis

Finally, prions , infectious agents composed only of protein, are

responsible for causing a variety of spongiform encephalopathies such as scrapie and “mad cow disease.”

1 Describe the fi eld of microbiology in terms of the size

of its subject material and the nature of its techniques

2 Describe and contrast procaryotic and eucaryotic cells

OF MICROBIOLOGY

As the scientist-writer Steven Jay Gould (1941–2002) emphasized,

we live in the age of bacteria They were the fi rst living organisms

on our planet, likely created the atmosphere that allowed the evolution of oxygen-consuming life-forms, and now live virtually everywhere life is possible Furthermore, the biosphere depends on their activities, and they infl uence human society in countless ways Because microorganisms play such diverse roles, modern microbiology is a large discipline with many different specialties;

it has a great impact on fi elds such as medicine, agricultural and food sciences, ecology, genetics, biochemistry, and molecular biology One indication of the importance of microbiology is the Nobel Prize given for work in physiology or medicine About one-third of these prizes have been awarded to scientists working

on microbiological problems ( see inside front cover )

Microbiology has both basic and applied aspects The basic aspects are concerned with the biology of microorganisms them-selves The applied aspects are concerned with practical problems such as disease, water and wastewater treatment, food spoilage

Figure 1.1 Universal Phylogenetic Tree These evolutionary relationships are based on rRNA sequence comparisons The human genus (Homo) is highlighted in red.

Methanothermus Methanopyrus

Thermofilum Thermoproteus Pyrodictium Sulfolobus

Methanospirillum

Haloferax Archaeoglobus Thermoplasma Methanococcus

Marine low temp

Coprinus

Zea Achlya Costaria Porphyra Paramecium Babesia

Dictyostelium Entamoeba Naegleria

Euglena T rypanosoma Physarum

Encephalitozoon V airimorpha

Trichomonas

Giardia

Cryptomonas

Methanobacterium Flavobacterium

Chloroflexus Thermus

Thermotoga Aquifex

pOPS66 EM17 pOPS19

Chloroplast

Eucarya

Archaea

Bacteria

Root Gp 3 low tempGp 2 low tempGp 1 low temp

Marine Gp 1 low temp

pJP 27 pJP 78

pSL 22 pSL 12 pSL 50

Homo

1.2 Scope and Relevance of Microbiology 3

4 Chapter 1 The History and Scope of Microbiology

and food production, and industrial uses of microbes It is

impor-tant to note that the basic and applied aspects of microbiology are

intertwined Basic research is often conducted in applied fi elds,

and applications often arise out of basic research A discussion of

some of the major fi elds of microbiology and the occupations

within them follows

Although pathogenic microbes are the minority, they garner

considerable interest Thus, one of the most active and important

fi elds in microbiology is medical microbiology, which deals with

diseases of humans and animals Medical microbiologists

iden-tify the agents causing infectious diseases and plan measures for

their control and elimination Frequently they are involved in

tracking down new, unidentifi ed pathogens such as the agent that

causes variant Creutzfeldt-Jakob disease (the human version of

“mad cow disease”), hantavirus, West Nile virus, and the virus

responsible for SARS These microbiologists also study the ways

in which microorganisms cause disease >> Microbial Diversity

& Ecology 24.1: SARS: Evolution of a virus

As noted earlier, major epidemics have regularly affected

human history The 1918 infl uenza pandemic is of particular

note; it killed more than 20 million people in about one year

Public health microbiology is concerned with the control and

spread of such communicable diseases Public health

microbiol-ogists and epidemiolmicrobiol-ogists monitor the amount of disease in

populations Based on their observations, they can detect

out-breaks and developing epidemics, and implement appropriate

control measures in response They also conduct surveillance for

new diseases as well as bioterrorism events Those public health

microbiologists working for local governments monitor

commu-nity food establishments and water supplies in an attempt to keep

them safe and free from infectious disease agents

Immunology is concerned with how the immune system

pro-tects the body from pathogens and the response of infectious

agents It is one of the fastest growing areas in science Much of

the growth began with the discovery of HIV, which specifi cally

targets cells of the immune system Immunology also deals with

health problems such as the nature and treatment of allergies and

autoimmune diseases such as rheumatoid arthritis >> Techniques

& Applications 29.1: Monoclonal antibody technology

Agricultural microbiology is concerned with the impact of

microorganisms on agriculture Microbes such as nitrogen-fi xing

bacteria play critical roles in the nitrogen cycle and affect soil

fer-tility Other microbes live in the digestive tracts of ruminants such

as cattle and break down the plant materials these animals ingest

There are also plant and animal pathogens that can have signifi cant

economic impacts if not controlled Agricultural microbiologists

work on methods to increase soil fertility and crop yields, study

rumen microorganisms in order to increase meat and milk

produc-tion, and try to combat plant and animal diseases Currently many

agricultural microbiologists are studying the use of bacterial and

viral insect pathogens as substitutes for chemical pesticides

Microbial ecology is concerned with the relationships

between microorganisms and the components of their living and

nonliving habitats Microbial ecologists study the global and

local contributions of microorganisms to the carbon, nitrogen,

and sulfur cycles, including the role of microbes in both the

production and removal of greenhouse gases such as carbon dioxide and methane The study of pollution effects on microor-ganisms also is important because of the impact these organisms have on the environment Microbial ecologists are employing microorganisms in bioremediation to reduce pollution The study

of the microbes normally associated with the human body has become a new frontier in microbial ecology

Numerous foods are made using microorganisms On the other hand, some microbes cause food spoilage or are pathogens spread

through food An excellent example of the latter is Escherichia coli

O157:H7, which in 2006 caused a widespread outbreak of ease when it contaminated a major source of spinach in the United States Scientists working in food and dairy microbiology con-tinue to explore the use of microbes in food production They also work to prevent microbial spoilage of food and the transmis-sion of food-borne diseases There is also considerable research

dis-on the use of microorganisms themselves as a nutrient source for livestock and humans >> Microbiology of food (chapter 34)

In 1929 Alexander Fleming discovered that the fungus

Penicillium produced what he called penicillin, the fi rst antibiotic

that could successfully control bacterial infections Although it took World War II for scientists to learn how to mass-produce it, scientists soon found other microorganisms capable of producing additional antibiotics as well as compounds such as citric acid, vitamin B12, and monosodium glutamate (MSG) Today, indus-trial microbiologists use microorganisms to make products such

as antibiotics, vaccines, steroids, alcohols and other solvents, vitamins, amino acids, and enzymes Industrial microbiologists identify microbes of use to industry They also utilize techniques

to improve production by microbes and devise systems for turing them and isolating the products they make

Microbes are metabolically diverse and can employ a wide variety of energy sources, including organic matter, inorganic molecules (e.g., H 2 and NH 3), and sunlight Microbiologists working in microbial physiology and biochemistry study many aspects of the biology of microorganisms, including their metabolic capabilities They may also study the synthesis of anti-biotics and toxins, the ways in which microorganisms survive harsh environmental conditions, and the effects of chemical and physical agents on microbial growth and survival

Microbial genetics and molecular biology focus on the nature

of genetic information and how it regulates the development and

function of cells and organisms The bacteria E coli and Bacillus subtilis, the yeast Saccharomyces cerevisiae (baker’s yeast), and

bacterial viruses such as T4 and lambda continue to be important model organisms used to understand biological phenomena

Microbial geneticists also play a signifi cant role in applied microbiology because they develop techniques that are useful in agricultural microbiology, industrial microbiology, food and dairy microbiology, and medicine

Because of the practical importance of microbes and their use

as model organisms, the future of microbiology is bright

However, it is important to remember that future advances in microbiology will build on the foundations laid by earlier scien-tists The development of microbiology as a science is described

in sections 1.3 to 1.5 Figure 1.2 presents a summary of some of

Figure 1.2 Some Important Events in the Development of Microbiology Milestones in microbiology are marked in red; other historical events are in black.

1798 Jenner introduces cowpox vaccination for smallpox.

1911 Rous discovers a virus can cause cancer.

1915-1917 D’Herelle and Twort discover bacterial viruses.

1923 First edition of

Bergey’s Manual

1928 Griffith discovers bacterial transformation.

1929 Fleming discovers penicillin.

1953 Watson and Crick propose DNA double helix.

1961 Jacob and Monod

propose lac operon.

1970 Arber and Smith discover restriction endonucleases.

1977 Woese divides procaryotes into

Bacteria and Archaea.

1900 Planck develops quantum theory.

1903 Wright brothers’

first powered aircraft

1905 Einstein’s theory of relativity

1908 First Model T Ford

1914 World War I begins 1917 Russian

Revolution

1927 Lindberg’s transAtlantic flight

1929 Stock market crash

1933 Hitler becomes chancellor

of Germany

1961 First human

in space

1969 Neil Armstrong walks on the moon.

1973 Vietnam War ends.

1980 First home computers

1937 Krebs discovers citric acid cycle.

1939 World War II begins.

1945 Atomic bomb dropped on Hiroshima.

1950 Korean War begins.

1983-1984 HIV isolated and identified by Gallo and Montagnier;

Mullis develops PCR technique.

1990 First human gene therapy testing begun.

1992 First human trials

of antisense therapy.

2001 Anthrax bioterrorism attacks in New York, Washington D.C., and Florida.

2003 SARS outbreak

in China

1981 First space shuttle launch

1991 Soviet Union collapses.

2001 World Trade Center attack

2003 Second war with Iraq

1590–1608 Jansen develops first useful compound microscope.

1665 Hooke publishes

Micrographia.

1676 Leeuwenhoek discovers “animacules.”

1688 Redi refutes spontaneous generation

of maggots.

1765–1776 Spallanzani attacks spontaneous generation.

1861 Pasteur disproves spontaneous generation.

1887-1890 Winogradsky studies sulfur and nitrifying bacteria.

1889 Beijerinck isolates root nodule bacteria.

1899 Beijerinck proves virus causes tobacco mosaic disease.

1884 Koch’s postulates published; Metchnikoff describes phagocytosis;

autoclave developed;

Gram stain developed.

1543 Publication of Copernicus’s work

on heliocentric solar system 1687 Newton’sPrincipia published 1776 AmericanRevolution

1859 Darwin’s

Origin of Species

1861–1865 American Civil War

1876 Bell invents telephone.

1898 American War

Spanish-1879 Edison’s first light bulb

6 Chapter 1 The History and Scope of Microbiology

the major events in this process and their relationship to other

historical landmarks

1 Briefl y describe the major subdisciplines in

micro biology

2 Why do you think microorganisms are useful to

biologists as experimental models?

3 List all the activities or businesses you can think of in

your community that directly depend on microbiology

MICROORGANISMS

Even before microorganisms were seen, some investigators

suspected their existence and responsibility for disease Among

others, the Roman philosopher Lucretius (about 98–55 bce) and

the physician Girolamo Fracastoro (1478–1553) suggested that

disease was caused by invisible living creatures The earliest

micro-scopic observations appear to have been made between 1625 and

1630 on bees and weevils by the Italian Francesco Stelluti, using a

microscope probably supplied by Galileo In 1665 the fi rst drawing

of a microorganism was published in Robert Hooke’s Micrographia

However, the fi rst person to publish extensive, accurate

observa-tions of microorganisms was the amateur microscopist Antony van

Leeuwenhoek (1632–1723) of Delft, the Netherlands ( fi gure 1.3 a )

Leeuwenhoek earned his living as a draper and haberdasher (a

dealer in men’s clothing and accessories) but spent much of his

spare time constructing simple microscopes composed of double

convex glass lenses held between two silver plates ( fi gure 1.3 b )

His microscopes could magnify around 50 to 300 times, and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45°

angle to the specimen plane This would have provided a form of dark-fi eld illumination in which the organisms appeared as bright objects against a dark background and made bacteria clearly visi-

ble ( fi gure 1.3 c ) Beginning in 1673, Leeuwenhoek sent detailed

letters describing his discoveries to the Royal Society of London

It is clear from his descriptions that he saw both procaryotes and protozoa

(c)

Lens Specimen holder

Focus screw

Handle

(b)

Figure 1.3 Antony van Leeuwenhoek (a) An oil painting of Leeuwenhoek (b) A brass replica of the Leeuwenhoek microscope Inset photo

shows how it is held (c) Leeuwenhoek’s drawings of bacteria from the human mouth.

(a)

As important as Leeuwenhoek’s observations were, the opment of microbiology essentially languished for the next

devel-200 years Little progress was made primarily because scopic observations of microorganisms do not provide suffi cient information to understand their biology For the discipline to develop, techniques for isolating and culturing microbes in the laboratory were needed Many of these techniques began to be developed as scientists grappled with the confl ict over the theory

micro-of spontaneous generation This confl ict and the subsequent studies

on the role played by microorganisms in causing disease ultimately led to what is now called the golden age of microbiology

1 Give some examples of the kind of information you think can be provided by microscopic observations of microorganisms

2 Give some examples of the kind of information you think can be provided by isolating microorganisms from their natural environment and culturing them in the laboratory

SPONTANEOUS GENERATION

From earliest times, people had believed in spontaneous generation —that living organisms could develop from nonliv-

ing matter Even Aristotle (384–322 bce) thought some of the simpler invertebrates could arise by spontaneous generation

This view fi nally was challenged by the Italian physician Francesco Redi (1626–1697), who carried out a series of experi-ments on decaying meat and its ability to produce maggots spontaneously Redi placed meat in three containers One was uncovered, a second was covered with paper, and the third was covered with fi ne gauze that would exclude fl ies Flies laid their eggs on the uncovered meat and maggots developed The other two pieces of meat did not produce maggots spontaneously

However, fl ies were attracted to the gauze-covered container and laid their eggs on the gauze; these eggs produced maggots Thus the generation of maggots by decaying meat resulted from the presence of fl y eggs, and meat did not spontaneously generate maggots as previously believed Similar experiments by others helped discredit the theory for larger organisms

Leeuwenhoek’s discovery of microorganisms renewed the controversy Some proposed that microorganisms arose by spon-taneous generation even though larger organisms did not They pointed out that boiled extracts of hay or meat gave rise to micro-organisms after sitting for a while In 1748 the English priest John Needham (1713–1781) reported the results of his experiments on spontaneous generation Needham boiled mutton broth in fl asks that he then tightly stoppered Eventually many of the fl asks became cloudy and contained microorganisms He thought organic matter contained a vital force that could confer the properties

of life on nonliving matter A few years later, the Italian priest and naturalist Lazzaro Spallanzani (1729–1799) improved on Needham’s experimental design by fi rst sealing glass fl asks that contained water and seeds If the sealed fl asks were placed in boil-ing water for three-quarters of an hour, no growth took place as long as the fl asks remained sealed He proposed that air carried germs to the culture medium but also commented that the external air might be required for growth of animals already in the medium

The supporters of spontaneous generation maintained that heating the air in sealed fl asks destroyed its ability to support life

Several investigators attempted to counter such arguments

Theodore Schwann (1810–1882) allowed air to enter a fl ask containing a sterile nutrient solution after the air had passed through a red-hot tube The fl ask remained sterile Subsequently Georg Friedrich Schroder (1810–1885) and Theodor von Dusch (1824–1890) allowed air to enter a fl ask of heat-sterilized medium after it had passed through sterile cotton wool No growth occurred in the medium even though the air had not been heated Despite these experiments, the French naturalist Felix Pouchet (1800–1872) claimed in 1859 to have carried out experiments conclusively proving that microbial growth could occur without air contamination This claim provoked Louis Pasteur (1822–1895) to settle the matter Pasteur ( fi gure 1.4 )

Figure 1.4 Louis Pasteur Pasteur working in his laboratory.

1.4 Conflict Over Spontaneous Generation 7

8 Chapter 1 The History and Scope of Microbiology

fi rst fi ltered air through cotton and found that objects

resem-bling plant spores had been trapped If a piece of the cotton

was placed in sterile medium after air had been fi ltered through

it, microbial growth occurred Next he placed nutrient

solu-tions in fl asks, heated their necks in a fl ame, and drew them out

into a variety of curves The swan neck fl asks that he produced

in this way had necks open to the atmosphere ( fi gure 1.5 )

Pasteur then boiled the solutions for a few minutes and allowed

them to cool No growth took place even though the contents

of the fl asks were exposed to the air Pasteur pointed out that

no growth occurred because dust and germs had been trapped

on the walls of the curved necks If the necks were broken,

growth commenced immediately Pasteur had not only resolved

the controversy by 1861 but also had shown how to keep

solu-tions sterile

The English physicist John Tyndall (1820–1893) and the

German botanist Ferdinand Cohn (1828–1898) dealt a fi nal

blow to spontaneous generation In 1877 Tyndall demonstrated

that dust did indeed carry germs and that if dust was absent,

broth remained sterile even if directly exposed to air During

the course of his studies, Tyndall provided evidence for the

existence of exceptionally heat-resistant forms of bacteria

Working independently, Cohn discovered that the heat- resistant

bacteria recognized by Tyndall were species capable of

pro-ducing bacterial endospores Cohn later played an instrumental

role in establishing a classifi cation system for procaryotes

based on their morphology and physiology >> Bacterial

MICROBIOLOGY

Pasteur’s work with swan neck fl asks ushered in the golden age

of microbiology Within 60 years (1857–1914), a number of disease-causing microbes were discovered, great strides in under-standing microbial metabolism were made, and techniques for isolating and characterizing microbes were improved Scientists also identifi ed the role of immunity in preventing disease and controlling microbes, developed vaccines, and introduced tech-niques used to prevent infection during surgery

Microorganisms and Disease

Although Fracastoro and a few others had suggested that invisible organisms produced disease, most people believed that disease was due to causes such as supernatural forces, poisonous vapors called miasmas, and imbalances among the four humors thought

to be present in the body The role of the four humors (blood, phlegm, yellow bile [choler], and black bile [melancholy]) in disease had been widely accepted since the time of the Greek phy-sician Galen (129–199) Support for the idea that microorganisms cause disease—that is, the germ theory of disease—began to accumulate in the early nineteenth century from diverse fi elds

Agostino Bassi (1773–1856) fi rst showed a microorganism could cause disease when he demonstrated in 1835 that a silkworm dis-ease was due to a fungal infection He also suggested that many diseases were due to microbial infections In 1845 M J Berkeley (1803–1889) proved that the great potato blight of Ireland was caused by a water mold, and in 1853 Heinrich de Bary (1831–1888) showed that smut and rust fungi caused cereal crop diseases Follow-ing his successes with the study of fermentation, Pasteur was asked

by the French government to investigate the pèbrine disease of

silk-worms that was disrupting the silk industry After several years of work, he showed that the disease was due to a protozoan parasite

Indirect evidence for the germ theory of disease came from the work of the English surgeon Joseph Lister (1827–1912) on the prevention of wound infections Lister, impressed with Pasteur’s studies on the involvement of microorganisms in fermentation and putrefaction, developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds Instruments were heat sterilized, and phenol was used

on surgical dressings and at times sprayed over the surgical area

The approach was remarkably successful and transformed surgery It also provided strong indirect evidence for the role of microorganisms in disease because phenol, which kills bacteria, also prevented wound infections

Figure 1.5 Pasteur’s Swan Neck Flasks These fl asks were

used in his experiments on the spontaneous generation of microorganisms

Source: Annales Sciences Naturelle 4th series, vol 16, pp 1–98, Pasteur, L., 1861,

“Mémoire sur les Corpuscules Organisés Qui Existent Dans L’Apmosphére: Examen de

la Doctrine des Générations Spontanées.”

establish the relationship between Bacillus anthracis and anthrax;

he published his fi ndings in 1876 Koch injected healthy mice with material from diseased animals, and the mice became ill

After transferring anthrax by inoculation through a series of 20 mice, he incubated a piece of spleen containing the anthrax bacil-lus in beef serum The bacilli grew, reproduced, and produced

endospores When the isolated bacilli or their spores were injected into healthy mice, anthrax developed His criteria for proving the causal relationship between a microorganism and a specifi c disease

are known as Koch’s postulates Koch’s proof that B anthracis

caused anthrax was independently confi rmed by Pasteur and his coworkers They discovered that after burial of dead animals, anthrax spores survived and were brought to the surface by earth-worms Healthy animals then ingested the spores and became ill

After completing his anthrax studies, Koch fully outlined his postulates in his work on the cause of tuberculosis ( table 1.1 ) In

1884 he reported that this disease was caused by the rod-shaped

bacterium Mycobacterium tuberculosis, and he was awarded the

Nobel Prize in Physiology or Medicine in 1905 for his work

Koch’s postulates were quickly adopted by others and used to connect many diseases to their causative agent However, their use is at times not feasible ( Disease 1.1 ) For instance, organisms

such as Mycobacterium leprae, the causative agent of leprosy,

cannot be isolated in pure culture

Development of Techniques for Studying Microbial Pathogens

During Koch’s studies on bacterial diseases, it became necessary

to isolate suspected bacterial pathogens in pure culture—a ture containing only one type of microorganism At fi rst, Koch cultured bacteria on the sterile surfaces of cut, boiled potatoes, but the bacteria would not always grow well Eventually he developed culture media using meat extracts and protein digests, reasoning these were similar to body fl uids Initially he tried to solidify the media by adding gelatin Separate bacterial colonies developed after the surface of the solidifi ed medium had been streaked with a bacterial sample The sample could also be mixed with liquefi ed gelatin medium When the gelatin medium hard-ened, individual bacteria produced separate colonies Despite its advantages, gelatin was not an ideal solidifying agent because it can be digested by many microbes and melts at temperatures

cul-Figure 1.6 Robert Koch Koch examining a specimen in his laboratory.

tuberculosis Is the Causative Agent of Tuberculosis

1 The microorganism must be present in every case of the disease but absent from healthy organisms.

Koch developed a staining technique to examine human tissue

M tuberculosis cells could be identifi ed in diseased tissue.

2 The suspected microorganisms must be isolated and grown

in a pure culture.

Koch grew M tuberculosis in pure culture on coagulated blood serum.

3 The same disease must result when the isolated microorganism is inoculated into a healthy host.

Koch injected cells from the pure culture of M tuberculosis into guinea

pigs The guinea pigs subsequently died of tuberculosis.

4 The same microorganism must be isolated again from the diseased host.

Koch isolated M tuberculosis from the dead guinea pigs and was able to

again culture the microbe in pure culture on coagulated blood serum.

Table 1.1

1.5 Golden Age of Microbiology 9

10 Chapter 1 The History and Scope of Microbiology

above 28°C A better alternative was provided by Fanny

Eilshemius Hesse (1850–1934), the wife of Walther Hesse

(1846–1911), one of Koch’s assistants She suggested the use of

agar as a solidifying agent, which she used to make jellies Agar

was not attacked by most bacteria Furthermore, it did not melt

until reaching a temperature of 100°C and, once melted, did not

solidify until reaching a temperature of 50°C; this eliminated the

need to handle boiling liquid Some of the media developed by

Koch and his associates, such as nutrient broth and nutrient agar,

are still widely used Another important tool developed in Koch’s

laboratory was a container for holding solidifi ed media—the

petri dish (plate), named after Richard Petri (1852–1921), who

devised it These developments directly stimulated progress in all

areas of microbiology >> Culture media (section 6.7); Isolation of

pure cultures (section 6.8)

Viral pathogens were also studied during this time The

dis-covery of viruses and their role in disease was made possible

when Charles Chamberland (1851–1908), one of Pasteur’s

asso-ciates, constructed a porcelain bacterial fi lter in 1884 Dimitri

Ivanowski (1864–1920) and Martinus Beijerinck (pronounced

“by-a-rink;” 1851–1931) used the fi lter to study tobacco

mosaic disease They found that plant extracts and sap from

diseased plants were infectious, even after being fi ltered with

Chamberland’s fi lter Because the infectious agent passed through

a fi lter that was designed to trap bacterial cells, they reasoned that

the agent must be something smaller than a bacterium Beijerinck

proposed that the agent was a “fi lterable virus.” Eventually

viruses were shown to be tiny, acellular infectious agents

Immunological Studies

In this period, progress also was made in determining how animals

resisted disease and in developing techniques for protecting

humans and livestock against pathogens During studies on chicken

cholera, Pasteur and Pierre Roux (1853–1933) discovered that

incubating the cultures for long intervals between transfers would attenuate the bacteria, which meant they had lost their ability to cause the disease If the chickens were injected with these attenuated cultures, they remained healthy and developed the ability to resist the disease when exposed to virulent cultures Pasteur called the

attenuated culture a vaccine (Latin vacca, cow) in honor of Edward

Jenner (1749–1823) because, many years earlier, Jenner had used material from cowpox lesions to protect people against smallpox

Shortly after this, Pasteur and Chamberland developed an attenuated anthrax vaccine >> Control of epidemics: Vaccines and immunizations (section 33.8)

Pasteur also prepared a rabies vaccine using an attenuated

strain of Rabies virus During the course of these studies, Joseph

Meister, a nine-year-old boy who had been bitten by a rabid dog, was brought to Pasteur Since the boy’s death was certain in the absence of treatment, Pasteur agreed to try vaccination Joseph was injected 13 times over the next 10 days with increasingly virulent preparations of the attenuated virus He survived In gratitude for Pasteur’s development of vaccines, people from around the world contributed to the construction of the Pasteur Institute in Paris, France One of the initial tasks of the institute was vaccine production

After the discovery that the diphtheria bacillus produced a toxin, Emil von Behring (1854–1917) and Shibasaburo Kitasato (1852–1931) injected inactivated toxin into rabbits, inducing them to produce an antitoxin, a substance in the blood that would inactivate the diphtheria toxin and protect against the disease A tetanus antitoxin was then prepared, and both antitoxins were used in the treatment of people

The antitoxin work provided evidence that immunity could result from soluble substances in the blood, now known to be antibodies (humoral immunity) It became clear that blood cells were also important in immunity (cellular immunity) when Elie Metchnikoff (1845–1916) discovered that some white blood cells could engulf disease-causing bacteria ( fi gure 1.7 ) He called

Disease

Although the criteria that Koch developed for proving a causal

relation-ship between a microorganism and a specifi c disease have been of great

importance in medical microbiology, it is not always possible to apply

them in studying human diseases For example, some pathogens cannot

be grown in pure culture outside the host; because other pathogens grow

only in humans, their study would require experimentation on people

The identifi cation, isolation, and cloning of genes responsible for

viru-lence have made possible a new molecular form of Koch’s postulates

that resolves some of these diffi culties The emphasis is on the virulence

genes present in the infectious agent rather than on the agent itself The

molecular postulates can be briefl y summarized as follows:

1 The virulence trait under study should be associated much

more with pathogenic strains of the species than with pathogenic strains.

2 Inactivation of the gene or genes associated with the pected virulence trait should substantially decrease pathogenicity.

sus-3 Replacement of the mutated gene with the normal type gene should fully restore pathogenicity.

wild-4 The gene should be expressed at some point during the infection and disease process.

5 Antibodies or immune system cells directed against the gene products should protect the host.

The molecular approach cannot always be applied because of lems such as the lack of an appropriate animal system It also is diffi cult to employ the molecular postulates when the pathogen is not well characterized genetically.

prob-wiL75233_ch01_001-012.indd Page 10 10/20/07 10:45:49 AM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12

these cells phagocytes and the process phagocytosis (Greek

phagein, eating)

1 Discuss the contributions of Lister, Pasteur, and Koch

to the germ theory of disease and the treatment or prevention of diseases

2 What other contributions did Koch make to microbiology?

3 Describe Koch’s postulates What is a pure culture?

Why are pure cultures important to Koch’s postulates?

4 Would microbiology have developed more slowly if Fanny Hesse had not suggested the use of agar? Give your reasoning

5 Some individuals can be infected by a pathogen yet not develop disease In fact, some become chronic carri-ers of the pathogen How does this observation affect Koch’s postulates? How might the postulates be modi-

fi ed to account for the existence of chronic carriers?

6 How did Jenner, von Behring, Kitasato, and nikoff contribute to the development of immunology?

OF INDUSTRIAL MICROBIOLOGY AND MICROBIAL ECOLOGY

Although humans had unknowingly exploited microbes for sands of years, industrial microbiology developed in large part from the work of Louis Pasteur and others on the alcoholic fermentations that yielded wine and other alcoholic beverages In

thou-1837, when Theodore Schwann and others proposed that yeast

cells were responsible for the conversion of sugars to alcohol, the leading chemists of the time believed microorganisms were not involved They were convinced that fermentation was due to a chemical instability that degraded the sugars to alcohol Pasteur did not agree; he believed that fermentations were carried out by living organisms In 1856 M Bigo, an industrialist in Lille, France, where Pasteur worked, requested Pasteur’s assistance His busi-ness produced ethanol from the fermentation of beet sugars, and the alcohol yields had recently declined and the product had become sour Pasteur discovered that the fermentation was failing because the yeast normally responsible for alcohol formation had been replaced by bacteria that produced acid rather than ethanol

In solving this practical problem, Pasteur demonstrated that all fermentations were due to the activities of specifi c yeasts and bac-teria, and he published several papers on fermentation between

1857 and 1860 His success led to a study of wine diseases and the development of pasteurization to preserve wine during storage

Pasteur’s studies on fermentation continued for almost 20 years

One of his most important discoveries was that some fermentative microorganisms were anaerobic and could live only in the absence

of oxygen, whereas others were able to live either aerobically or anaerobically >> Controlling food spoilage (section 34.3)

Microbial ecology developed when a few of the early ologists chose to investigate the ecological role of microorganisms

microbi-In particular, they studied microbial involvement in the carbon, nitrogen, and sulfur cycles taking place in soil and aquatic habitats

The Russian microbiologist Sergei Winogradsky (1856–1953) made many contributions to soil microbiology He discovered that soil bacteria could oxidize iron, sulfur, and ammonia to obtain energy and that many of these bacteria could incorporate CO 2 into organic matter much as photosynthetic organisms do Winogradsky also isolated anaerobic nitrogen-fi xing soil bacteria and studied the decomposition of cellulose Martinus Beijerinck was one of the great general microbiologists who made fundamental contribu-tions to microbial ecology and many other fi elds He isolated the

aerobic nitrogen-fi xing bacterium Azotobacter, a root nodule terium also capable of fi xing nitrogen (later named Rhizobium ),

bac-and sulfate-reducing bacteria Beijerinck bac-and Winogradsky also developed the enrichment-culture technique and the use of selec-tive media, which have been of great importance in microbiology

>> Biogeochemical cycling (section 25.1); Culture media (section 6.7)

1 Briefl y describe Pasteur’s work on microbial tions

2 How did Winogradsky and Beijerinck contribute to the study of microbial ecology?

3 Leeuwenhoek is often referred to as the father of crobiology However, many historians feel that Louis Pasteur, Robert Koch, or perhaps both, deserve that honor Who do you think is the father of microbiology?

mi-Why?

4 Consider the discoveries described in sections 1.3 to 1.6 Which do you think were the most important to the development of microbiology? Why?

Figure 1.7 Elie Metchnikoff Metchnikoff at work in his laboratory.

1.6 Development of Industrial Microbiology and Microbial Ecology 11

Critical Thinking Questions

1 Consider the impact of microbes on the course of world history

History is full of examples of instances or circumstances under

which one group of people lost a struggle against another In

fact, when examined more closely, the “losers” often had the

misfortune of being exposed to, more susceptible to, or unable

to cope with an infectious agent Thus weakened in physical

strength or demoralized by the course of a devastating disease,

they were easily overcome by human “conquerors.”

a Choose an example of a battle or other human activity such

as exploration of new territory and determine the impact

of microorganisms, either indigenous or transported to the region, on that activity.

b Discuss the effect that the microbe(s) had on the outcome

in your example.

c Suggest whether the advent of antibiotics, food storage and

preparation technology, or sterilization technology would have made a difference in the outcome.

2 Vaccinations against various childhood diseases have contributed

to the entry of women, particularly mothers, into the full-time workplace.

a Is this statement supported by data comparing availability and extent of vaccination with employment statistics in dif- ferent places or at different times?

b Before vaccinations for measles, mumps, and chickenpox, what was the incubation time and duration of these child- hood diseases? What impact would such diseases have on mothers with several elementary schoolchildren at home

if they had full-time jobs and lacked substantial child care support?

c What would be the consequence if an entire generation

of children (or a group of children in one country) was not vaccinated against any diseases? What do you predict would happen if these children went to college and lived

in a dormitory in close proximity with others who had received all of the recommended childhood vaccines?

Summary

a Microbiology studies microscopic organisms that are often

unicellular or, if multicellular, do not have highly differentiated

tissues The discipline is also defi ned by the techniques it uses—in

particular, those used to isolate and culture microorganisms

b Procaryotic cells differ from eucaryotic cells in their lack of a

membrane-delimited nucleus and other ways as well

c Microbiologists divide organisms into three domains: Bacteria,

Archaea, and Eucarya

d Domains Bacteria and Archaea consist of procaryotic

microor-ganisms The eucaryotic microbes (protists and fungi) are placed

in Eucarya Viruses, viroids, and virusoids are acellular entities

that are not placed in any of the domains but are classifi ed by a

separate system

1.2 Scope and Relevance of Microbiology

a Microbiology has contributed greatly to the fi elds of medicine,

genetics, agriculture, food science, biochemistry, and molecular

biology

b There are many fi elds in microbiology These include the more

applied disciplines such as medical, public health, industrial, and

food and dairy microbiology Microbial ecology, physiology,

biochemistry, and genetics are examples of basic

microbiologi-cal research fi elds

a Antony van Leeuwenhoek was the fi rst person to extensively

describe microorganisms

1.4 Confl ict over Spontaneous Generation

a Experiments by Redi and others disproved the theory of neous generation of larger organisms

b The spontaneous generation of microorganisms was disproved

by Spallanzani, Pasteur, Tyndall, Cohn, and others

1.5 Golden Age of Microbiology

a Support for the germ theory of disease came from the work of Bassi, Pasteur, Koch, and others Lister provided indirect evi- dence with his development of antiseptic surgery

b Koch’s postulates are used to prove a direct relationship between

a suspected pathogen and a disease

c Koch and his coworkers developed the techniques required to grow bacteria on solid media and to isolate pure cultures of pathogens

d Vaccines against anthrax and rabies were made by Pasteur; von Behring and Kitasato prepared antitoxins for diphtheria and tetanus

e Metchnikoff discovered some white blood cells could tose and destroy bacterial pathogens

Microbial Ecology

a Pasteur showed that fermentations were caused by isms and that some microorganisms could live in the absence of oxygen

b The role of microorganisms in carbon, nitrogen, and sulfur cycles was fi rst studied by Winogradsky and Beijerinck

Learn More

12 Chapter 1 The History and Scope of Microbiology

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There are more animals living in the scum on the teeth in a man’s mouth

than there are men in a whole kingdom

— Antony van Leeuwenhoek

Chapter Glossary

Microscopes and the Study of Microbial

Structure

bright-fi eld microscope A microscope that illuminates the

specimen directly with bright light and forms a dark image on

a brighter background

confocal scanning laser microscope (CSLM) A light

micro-scope in which laser-derived light scans across the men at a specifi c level and illuminates one area at a time;

speci-stray light from other parts of the specimen is blocked out to give an image with excellent contrast and resolution

dark-fi eld microscope A microscope that brightly illuminates

the specimen while leaving the background dark

differential interference contrast (DIC) microscopy A type

of microscopy that combines two beams of plane polarized light after passing through a specimen; their interference is used to create the image

differential staining Staining procedures that divide microbes

into separate groups based on staining properties

fi xation The process in which the internal and external

structures of cells and organisms are preserved and fi xed in position

fl uorescence microscopy A type of microscopy that exposes

a specimen to light of a specifi c wavelength and then forms an image from the fl uorescent light produced; usually the speci- men is stained with a fl uorescent dye (fl uorochrome)

Gram stain A differential staining procedure that divides

bac-teria into gram-positive and gram-negative groups based on their ability to retain crystal violet when decolorized with an organic solvent such as ethanol

negative staining A staining procedure in which a dye is used

to make the background dark while the specimen is unstained

parfocal A microscope that retains proper focus when the

objectives are changed

phase-contrast microscope A microscope that converts

slight differences in refractive index and cell density into easily observed differences in light intensity

refractive index A measure of how much a substance

defl ects a light ray from a straight path as it passes from one medium (e.g., glass) to another (e.g., air)

resolution The ability of a microscope to separate or

distin-guish between small objects that are close together

scanning electron microscope (SEM) An electron

micro-scope that scans a beam of electrons over the surface of a specimen and forms an image of the surface from the elec- trons that are emitted by it

scanning probe microscope A microscope used to study

surface features by moving a sharp probe over the object’s surface (e.g., the scanning tunneling microscope)

simple staining Staining a specimen with a single dye

transmission electron microscope (TEM) A microscope in

which an image is formed by passing an electron beam through a specimen and focusing the scattered electrons with magnetic lenses

13

Clostridium tenani is a rod-shaped bacterium that forms endospores

and releases tetanus toxin, the cause of tetanus In this colorized phase-contrast micrograph, the endospores are the bright, oval objects located at the ends of the rods

14 Chapter 2 Microscopes and the Study of Microbial Structure

BENDING OF LIGHT

To understand how a light microscope operates, one must know

something about the way lenses bend and focus light to form

images When a ray of light passes from one medium to another,

refraction occurs—that is, the ray is bent at the interface The

refractive index is a measure of how greatly a substance slows

the velocity of light; the direction and magnitude of bending are

determined by the refractive indices of the two media forming

the interface For example, when light passes from air into

glass, a medium with a greater refractive index, it is slowed and

bent toward the normal, a line perpendicular to the surface

( fi gure 2.1 ) As light leaves glass and returns to air, a medium

with a lower refractive index, it accelerates and is bent away

from the normal Thus a prism bends light because glass has a

different refractive index from air, and the light strikes its

sur-face at an angle

Lenses act like a collection of prisms operating as a unit

When the light source is distant so that parallel rays of light

strike the lens, a convex lens focuses these rays at a specifi c

point, the focal point ( F in fi gure 2.2 ) The distance between the

center of the lens and the focal point is called the focal length

( f in fi gure 2.2 )

Our eyes cannot focus on objects nearer than about 25 cm or

10 inches This limitation may be overcome by using a convex lens as a simple magnifi er (or microscope) and holding it close

to an object A magnifying glass provides a clear image at much closer range, and the object appears larger Lens strength is related to focal length; a lens with a short focal length magnifi es

an object more than a weaker lens having a longer focal length

1 Defi ne refraction, refractive index, focal point, and focal length

2 Describe the path of a light ray through a prism or lens

3 How is lens strength related to focal length? How do you think this principle is applied to corrective eyeglasses?

Microbiologists currently employ a variety of light scopes in their work; bright-fi eld, dark-fi eld, phase-contrast, and fl uorescence microscopes are most commonly used Each

micro-is useful for certain applications Modern microscopes are all

Microbiology usually is concerned with organisms so small they

cannot be seen distinctly with the unaided eye The organisms and

entities studied by microbiologists typically range in size from viruses,

which are measured in nanometers (nm), to protists, the largest of

which are about 200 micrometers (μm) in diameter ( table 2.1 ) Thus,

the microscope is of crucial importance, and it is important to

understand how microscopes work and how specimens are

pre-pared for examination

In this chapter we begin with a detailed treatment of the

standard bright-fi eld microscope and then describe other

com-mon types of light microscopes Next we discuss preparation and

staining of specimens for examination with the light microscope

This is followed by a description of transmission and scanning

electron microscopes, both of which are used extensively in current

microbiological research We close the chapter with a brief

intro-duction to two newer forms of microscopy: confocal microscopy

and scanning probe microscopy

Figure 2.1 The Bending of Light by a Prism Normals (lines perpendicular to the surface of the prism) are indicated by dashed lines As light enters the glass, it is bent toward the fi rst normal When light leaves the glass and returns to air, it is bent away from the second normal As a result the prism bends light passing through it

Figure 2.2 Lens Function A lens functions somewhat like a collection of prisms Light rays from a distant source are focused at the focal point F The focal point lies a distance f, the focal length, from the

lens center

f F

Common Units of Measurement

Unit Abbreviation Value

1 centimeter cm 10 −2 meter or 0.394 inches

compound microscopes That is, the magnifi ed image formed

by the objective lens (the lens closest to the object being

examined) is further enlarged by one or more additional lenses

Bright-Field Microscope The bright-fi eld microscope is routinely used in microbiology

labs, where it can be employed to examine both stained and unstained specimens It is called a bright-fi eld microscope because it forms a dark image against a brighter background It consists of a sturdy metal body or stand composed of a base and

an arm to which the remaining parts are attached ( fi gure 2.3 ) A light source, either a mirror or an electric illuminator, is located

in the base Two focusing knobs, the fi ne and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image

The stage is positioned about halfway up the arm It holds microscope slides either by simple slide clips or by a mechanical stage clip A mechanical stage allows the operator to move a slide

smoothly during viewing by use of stage control knobs The stage condenser (or simply condenser) is mounted within or

sub-beneath the stage and focuses a cone of light on the slide Its tion often is fi xed in simpler microscopes but can be adjusted vertically in more advanced models

Figure 2.3 A Bright-Field Microscope The parts of a modern bright-fi eld microscope The microscope pictured is somewhat more cated than those found in many student laboratories For example, it is binocular (has two eyepieces) and has a mechanical stage, an adjustable substage condenser, and a built-in illuminator

sophisti-Ocular (eyepiece)

Body

Arm

Coarse focus adjustment knob Fine focus adjustment knob Stage adjustment knobs

Interpupillary adjustment

Nosepiece

Objective lens (4) Mechanical stage

Substage condenser Aperture diaphragm control Base with light source Field diaphragm lever

Light intensity control

The curved upper part of the arm holds the body assembly,

to which a nosepiece and one or more eyepieces or ocular lenses

are attached More advanced microscopes have eyepieces for both eyes and are called binocular microscopes The body assembly itself contains a series of mirrors and prisms so that the barrel holding the eyepiece may be tilted for ease in viewing

The nosepiece holds three to fi ve objective lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly Ideally a microscope should be

parfocal —that is, the image should remain in focus when objectives are changed

The image one sees when viewing a specimen with a pound microscope is created by the objective and ocular lenses working together Light from the illuminated specimen is focused

com-by the objective lens, creating an enlarged image within the microscope The ocular lens further magnifi es this primary image

The total magnifi cation is calculated by multiplying the objective and eyepiece magnifi cations together For example, if a 45 objective is used with a 10 eyepiece, the overall magnifi cation

16 Chapter 2 Microscopes and the Study of Microbial Structure

lens to separate or distinguish between small objects that are

close together

Resolution is described mathematically by an equation

devel-oped in the 1870s by Ernst Abbé, a German physicist responsible

for much of the optical theory underlying microscope design

The Abbé equation states that the minimal distance ( d ) between

two objects that reveals them as separate entities depends on the

wavelength of light (λ) used to illuminate the specimen and on

the numerical aperture of the lens ( n sin θ ), which is the ability

of the lens to gather light

d = 0.5 n sin λθ

As d becomes smaller, the resolution increases, and fi ner detail

can be discerned in a specimen; d becomes smaller as the

wave-length of light used decreases and as the numerical aperture (NA)

increases Thus the greatest resolution is obtained using a lens

with the largest possible NA and light of the shortest wavelength,

light at the blue end of the visible spectrum (in the range of 450

to 500 nm; see fi gure 7.24 )

The numerical aperture ( n sin θ) of a lens is defi ned by two

components: n is the refractive index of the medium in which

the lens works (e.g., air) and θ is 1/2 the angle of the cone of

light entering an objective ( fi gure 2.4 ) When this cone has a

narrow angle and tapers to a sharp point, it does not spread out much after leaving the slide and therefore does not adequately separate images of closely packed objects If the cone of light has a very wide angle and spreads out rapidly after passing through a specimen, closely packed objects appear widely sep-arated and are resolved The angle of the cone of light that can

enter a lens depends on the refractive index ( n ) of the medium

in which the lens works, as well as on the objective itself The refractive index for air is 1.00 and sin θ cannot be greater than

1 (the maximum θ is 90° and sin 90° is 1.00) Therefore no lens working in air can have a numerical aperture greater than 1.00

The only practical way to raise the numerical aperture above 1.00, and therefore achieve higher resolution, is to increase the refractive index with immersion oil, a colorless liquid with the

same refractive index as glass ( table 2.2 ) If air is replaced with

immersion oil, many light rays that did not enter the objective due to refl ection and refraction at the surfaces of the objective

lens and slide will now do so ( fi gure 2.5 ) This results in an

increase in numerical aperture and resolution

Numerical aperture is related to another characteristic of an objective lens, the working distance The working distance of an objective is the distance between the front surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus ( fi gure 2.4 ) Objectives with large numerical apertures and great resolving power have short work-ing distances ( table 2.2 )

Figure 2.4 Numerical Aperture in Microscopy The angular

aperture θ is 1/2 the angle of the cone of light that enters a lens from a

speci-men, and the numerical aperture is n sin θ In the right-hand illustration the

lens has larger angular and numerical apertures; its resolution is greater and

its working distance smaller

glass Slide

Figure 2.5 The Oil Immersion Objective An oil immersion objective operating in air and with immersion oil

The Properties of Microscope Objectives

Approximate resolving power

Table 2.2

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The preceding discussion has focused on the resolving power

of the objective lens The resolution of an entire microscope must take into account the numerical aperture of its condenser, as is evident from the following equation

Although the resolution of the microscope must consider both the condenser and the objective lens, in most cases the limit

of resolution of a light microscope is calculated using the Abbé equation, which considers the objective lens only The maximum theoretical resolving power of a microscope with an oil immer-sion objective (numerical aperture of 1.25) and blue-green light

10⫻ eyepieces and have an upper limit of about 1,000⫻ with oil immersion A 15⫻ eyepiece may be used with good objectives to achieve a useful magnifi cation of 1,500⫻ Any further magnifi -cation does not enable a person to see more detail Indeed, a light microscope can be built to yield a fi nal magnifi cation of 10,000⫻, but it would simply be magnifying a blur Only the electron microscope provides suffi cient resolution to make higher magni-

fi cations useful

Dark-Field Microscope

Although unpigmented living cells can be viewed in the

bright-fi eld microscope, they are not clearly visible because there is little difference in contrast between the cells and water As discussed

in section 2.3 , one solution to this problem is to kill and stain cells before observation to increase contrast and create variations

in color between cell structures But what if an investigator must view living cells to observe a dynamic process such as movement

or phagocytosis? Three types of light microscopes create detailed, clear images of living specimens: the dark-fi eld microscope, the phase-contrast microscope, and the differential interference con-trast microscope

The dark-fi eld microscope produces much more detailed

images of living, unstained cells and organisms by simply ing the way in which they are illuminated A hollow cone of light is focused on the specimen in such a way that unrefl ected and unrefracted rays do not enter the objective Only light that has been refl ected or refracted by the specimen forms an image ( fi gure 2.6 ) The fi eld surrounding a specimen appears black, while the object itself is brightly illuminated ( fi gure 2.7 a, b )

chang-The dark-fi eld microscope can reveal considerable internal

structure in larger eucaryotic microorganisms ( fi gure 2.7 b ) It

also is used to identify certain bacteria such as the thin and

dis-tinctively shaped Treponema pallidum ( fi gure 2.7 a ), the causative

agent of syphilis

Phase-Contrast Microscope

Dark-fi eld microscopy is one solution to viewing unpigmented

liv-ing cells Phase-contrast microscopy is another A phase-contrast microscope converts slight differences in refractive index and cell

density into easily detected variations in light intensity It is an

excellent way to observe living cells ( fi gure 2.7 c–e )

The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring, which produces

a hollow cone of light ( fi gure 2.8 ) As this cone passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen and are retarded by about 1/4 wavelength The deviated light is focused to form an image of the

(a)

(b)

Dark-field stop

Abbé condenser

Specimen Objective

Figure 2.6 Dark-Field Microscopy The simplest way to vert a microscope to dark-fi eld microscopy is to place (a) a dark-fi eld stop underneath (b) the condenser lens system The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen

con-2.2 Light Microscopes 17

18 Chapter 2 Microscopes and the Study of Microbial Structure

Figure 2.7 Examples of Dark-Field and Phase-Contrast

syphi-lis; dark-fi eld microscopy (b) Volvox and Spirogyra; dark-fi eld microscopy

(175) Note daughter colonies within the mature Volvox colony (center)

and the spiral chloroplasts of Spirogyra (left and right) (c) A phase-contrast

micrograph of Pseudomonas cells, which range from 1–3 μm in length

(d) Desulfotomaculum acetoxidans with spherical endospores and oblong

gas vacuoles; phase contrast (2,000) (e) Paramecium stained to show a

large central macronucleus with a small spherical micronucleus at its side;

phase-contrast microscopy ( 100)

(a) T pallidum: dark-fi eld microscopy

(b) Volvox and Spirogyra: dark-fi eld microscopy

(c) Pseudomonas: phase-contrast microscopy

(d) Desulfotomaculum: phase-contrast microscopy using a color fi lter

Micronucleus Macronucleus

(e) Paramecium: phase-contrast microscopy with stained specimen

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object Undeviated light rays strike a phase ring in the phase plate, a special optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate

If the phase ring is constructed in such a way that the undeviated light passing through it is advanced by 1/4 wavelength, the devi-ated and undeviated waves will be about 1/2 wavelength out of phase and will cancel each other when they come together to form an image ( fi gure 2.9 ) The background, formed by unde-viated light, is bright, while the unstained object appears dark

Figure 2.8 Phase-Contrast Microscopy The optics of a dark-phase-contrast microscope

Dark image with bright background results

of light pass through phase plate unchanged because they miss the phase ring.

Diffracted rays are retarded 1/4 wavelength after passing through objects.

Annular stop

Condenser

Direct light rays are advanced 1/4 wavelength as they pass through the phase ring.

and well defi ned This type of microscopy is called contrast microscopy Color fi lters often are used to improve the

dark-phase-image ( fi gure 2.7 d )

Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial components such as endospores and inclusion bodies that contain poly-β-hydroxyalkanoates (e.g., poly-β-hydroxybutyrate), polymetaphosphate, sulfur, or other substances

These are clearly visible ( fi gure 2.7 d ) because they have refractive

indices markedly different from that of water Phase-contrast microscopes also are widely used in studying eucaryotic cells >> Procaryotic cytoplasm: Inclusion bodies ( section 3.3 )

Differential Interference Contrast Microscope

The differential interference contrast (DIC) microscope is

similar to the phase-contrast microscope in that it creates an image by detecting differences in refractive indices and thick-ness Two beams of plane-polarized light at right angles to each other are generated by prisms In one design, the object beam passes through the specimen, while the reference beam passes through a clear area of the slide After passing through the spec-imen, the two beams are combined and interfere with each other

to form an image A live, unstained specimen appears brightly colored and three-dimensional ( fi gure 2.10 ) Structures such as cell walls, endospores, granules, vacuoles, and eucaryotic nuclei are clearly visible

Fluorescence Microscope

The microscopes thus far considered produce an image from light that passes through a specimen An object also can be seen because it actually emits light: this is the basis of fl uorescence microscopy When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light

Any light emitted by an excited molecule will have a longer wavelength (or be of lower energy) than the radiation originally absorbed Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state

The fl uorescence microscope exposes a specimen to

ultra-violet, ultra-violet, or blue light and forms an image of the object with the resulting fl uorescent light The most commonly used

fl uorescence microscopy is epifl uorescence microscopy, also called incident light or refl ected light fl uorescence micros-copy Epifl uorescence microscopes employ an objective lens that also acts as a condenser ( fi gure 2.11 ) A mercury vapor arc lamp or other source produces an intense beam of light that passes through an exciter fi lter The exciter fi lter transmits only the desired wavelength of excitation light The excitation light is directed down the microscope by a special mirror called the dichromatic mirror This mirror refl ects light of shorter wavelengths (i.e., the excitation light) but allows light of longer

2.2 Light Microscopes 19

20 Chapter 2 Microscopes and the Study of Microbial Structure

Bacterium Ray deviated by

specimen is 1/4 wavelength out

of phase.

Deviated ray is 1/2 wavelength out of phase.

Deviated and undeviated rays cancel each other out.

Phase plate

Figure 2.9 The Production of Contrast in Phase Microscopy The behavior of deviated and undeviated or undiffracted light rays in the dark-phase-contrast microscope Because the light rays tend to cancel each other out, the image of the specimen will be dark against a brighter background

Figure 2.10 Differential Interference Contrast Microscopy.

A micrograph of the protozoan Amoeba proteus The three-dimensional

image contains considerable detail and is artifi cially colored ( ⫻160)

fl uorescently labeled antibodies using immunofl uorescence cedures In ecological studies, the fl uorescence microscope is used to observe microorganisms stained with fl uorochrome-labeled probes or fl uorochromes that bind specifi c cell constituents ( table 2.3 ) In addition, microbial ecologists use epifl uo rescence microscopy to visualize photosynthetic microbes, as their pig-ments naturally fl uoresce when excited by light of specifi c wave-lengths It is even possible to distinguish live bacteria from dead bacteria by the color they fl uoresce after treatment with a specifi c mixture of stains ( fi gure 2.12 a ) Thus the microorganisms can be viewed and directly counted in a relatively undisturbed ecological niche >> Identifi cation of microorganisms from specimens: Micro- scopy ( section 32.2 )

pro-wavelengths to pass through The excitation light continues

down, passing through the objective lens to the specimen,

which is usually stained with molecules called fl uorochromes

( table 2.3 ) The fl uorochrome absorbs light energy from the

excitation light and fl uoresces brightly The emitted fl

uores-cent light travels up through the objective lens into the

micro-scope Because the emitted fl uorescent light has a longer

wavelength, it passes through the dichromatic mirror to a

bar-rier fi lter, which blocks out any residual excitation light

Finally, the emitted light passes through the barrier fi lter to

the eyepieces

The fl uorescence microscope has become an essential tool

in microbiology Bacterial pathogens (e.g., Mycobacterium

tuberculosis, the cause of tuberculosis) can be identifi ed after

staining with fl uorochromes or specifi cally tagging them with

Long wavelengths

Long wavelengths Short

wavelengths

Barrier filter (blocks ultraviolet radiation but allows visible light through) Dichromatic mirror reflects short wavelengths; transmits longer wavelengths.

Fluorochrome-coated specimen (absorbs short-wavelength radiation and emits longer-wavelength light)

Mercury arc lamp

Exciter filter (removes long wavelengths)

Figure 2.11 Epifl uorescence Microscopy The principles of operation of an epifl uorescence microscope

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1 List the parts of a light microscope and describe their functions

2 Defi ne resolution, numerical aperture, working tance, and fl uorochrome

3 If a specimen is viewed using a 5 objective in a microscope with a 15 eyepiece, how many times has the image been magnifi ed?

4 How does resolution depend on the wavelength of light, refractive index, and numerical aperture? How are resolution and magnifi cation related?

5 What is the function of immersion oil?

6 Why don’t most light microscopes use 30 ocular lenses for greater magnifi cation?

7 Briefl y describe how dark-fi eld, phase-contrast, ferential interference contrast, and epifl uorescence microscopes work and the kind of image provided by each Give a specifi c use for each type

AND STAINING OF SPECIMENS

Although living microorganisms can be directly examined with the light microscope, they often are fi xed and stained Such preparation serves to increase the visibility of the microorgan-isms, accentuate specifi c morphological features, and preserve them for future study

Fixation

The stained cells seen in a microscope should resemble living cells

as closely as possible Fixation is the process by which the internal

and external structures of cells and microorganisms are preserved and fi xed in position It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation A microorganism usually is killed and attached fi rmly to the microscope slide during fi xation

Myco-that is unique to Streptococcus pyogenes

2.3 Preparation and Staining of Specimens 21

Commonly Used Fluorochromes

Fluorescein isothiocyanate (FITC) Often attached to antibodies that bind specifi c cellular components or to DNA probes; fl uoresces greenTetramethyl rhodamine isothiocyanate

(TRITC or rhodamine)

Often attached to antibodies that bind specifi c cellular components;

fl uoresces red

Table 2.3

22 Chapter 2 Microscopes and the Study of Microbial Structure

There are two fundamentally different types of fi xation Heat

fi xation is routinely used to observe procaryotes Typically, a

fi lm of cells (a smear) is gently heated as a slide is passed through

a fl ame Heat fi xation preserves overall morphology but not

structures within cells Chemical fi xation is used to protect fi ne

cellular substructure and the morphology of larger, more delicate

microorganisms Chemical fi xatives penetrate cells and react

with cellular components, usually proteins and lipids, to render

them inactive, insoluble, and immobile Common fi xative

mix-tures contain such components as ethanol, acetic acid, mercuric

chloride, formaldehyde, and glutaraldehyde

Dyes and Simple Staining

The many types of dyes used to stain microorganisms have two

features in common: they have chromophore groups , groups

with conjugated double bonds that give the dye its color, and

they can bind with cells by ionic, covalent, or hydrophobic

bond-ing Most dyes are used to directly stain the cell or object of

interest, but some dyes (e.g., India ink and nigrosin) are used in

negative staining , where the background but not the cell is

stained; the unstained cells appear as bright objects against a

dark background

Dyes that bind cells by ionic interactions are probably the

most commonly used dyes These ionizable dyes may be

divided into two general classes based on the nature of their

charged group

1 Basic dyes —methylene blue, basic fuchsin, crystal

violet, safranin, malachite green—have positively

charged groups (usually some form of pentavalent

nitrogen) and are generally sold as chloride salts Basic

dyes bind to negatively charged molecules such as

nucleic acids, many proteins, and the surfaces of

procaryotic cells

2 Acidic dyes —eosin, rose bengal, and acid fuchsin—

possess negatively charged groups such as carboxyls

(—COOH) and phenolic hydroxyls (—OH) Acidic dyes,

because of their negative charge, bind to positively

charged cell structures

The staining effectiveness of ionizable dyes may be altered

by pH, since the nature and degree of the charge on cell

compo-nents change with pH Thus acidic dyes stain best under acidic

conditions when proteins and many other molecules carry a

posi-tive charge; basic dyes are most effecposi-tive at higher pHs

Dyes that bind through covalent bonds or because of their

solubility characteristics are also useful For instance, DNA can

be stained by the Feulgen procedure in which the staining

com-pound (Schiff’s reagent) is covalently attached to its deoxyribose

sugars Sudan III (Sudan Black) selectively stains lipids because

it is lipid soluble but does not dissolve in aqueous portions of

the cell

Microorganisms often can be stained very satisfactorily by

simple staining , in which a single dye is used ( fi gure 2.13 a,b )

Simple staining’s value lies in its simplicity and ease of use One covers the fi xed smear with stain for a short period, washes the excess stain off with water, and blots the slide dry Basic dyes such as crystal violet, methylene blue, and carbolfuchsin are fre-quently used in simple staining to determine the size, shape, and arrangement of procaryotic cells

Differential Staining The Gram stain , developed in 1884 by the Danish physician

Christian Gram, is the most widely employed staining method

in bacteriology It is an example of differential staining —

procedures that are used to distinguish organisms based on their

staining properties Use of the Gram stain divides Bacteria into

two groups—gram negative and gram positive

The Gram-staining procedure is illustrated in fi gure 2.14

In the fi rst step, the smear is stained with the basic dye crystal violet, the primary stain This is followed by treatment with an

iodine solution functioning as a mordant , a substance that

helps bind the dye to a cell Iodine increases the interaction between the cell and the dye so that the cell is stained more strongly The smear is next decolorized by washing with etha-nol or acetone This step generates the differential aspect of the Gram stain; gram-positive bacteria retain the crystal violet, whereas gram-negative bacteria lose the crystal violet and become colorless Finally, the smear is counterstained with a simple, basic dye different in color from crystal violet Safranin, the most common counterstain, colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple ( fi gures

2.13 c and 2.14 b ). >> Bacterial cell walls ( section 3.4 )

Acid-fast staining is another important differential staining

procedure It is most commonly used to identify Mycobacterium tuberculosis and M leprae ( fi gure 2.13 d ), the pathogens

responsible for tuberculosis and leprosy, respectively These bacteria have cell walls with high lipid content, in particular mycolic acids—a group of branched-chain hydroxy lipids, which prevent dyes from readily binding to the cells However,

M tuberculosis and M leprae can be stained by harsh

proce-dures such as the Ziehl-Neelsen method, which uses heat and phenol to drive basic fuchsin into cells Once basic fuchsin

has penetrated, M tuberculosis and M leprae are not easily

decolorized by acidifi ed alcohol (acid-alcohol) and thus are said to be acid-fast Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain

Staining Specifi c Structures

Many special staining procedures have been developed to study

specifi c structures with the light microscope Endospore ing , like acid-fast staining, also requires harsh treatment to

stain-drive dye into a target, in this case an endospore An endospore

is an exceptionally resistant structure produced by some

bacte-rial genera (e.g., Bacillus and Clostridium ) It can survive for

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2.3 Preparation and Staining of Specimens 23

Figure 2.13 Types of Microbiological Stains

long periods in an unfavorable environment, and it is called an endospore because it develops within the parent bacterial cell

Endospore morphology and location vary with species and often are valuable in identifi cation; endospores may be spherical to ellip-tical and either smaller or larger than the diameter of the parent

bacterium Endospores are not stained well by most dyes, but once stained they strongly resist decolorization This property is

the basis of most endospore staining methods ( fi gure 2.13 e ) In

the Schaeffer-Fulton procedure, endospores are fi rst stained by heating bacteria with malachite green, which is a very strong stain

Simple Stains

Differential Stains

Special Stains

(c) (a) (b)

Red cells are gram negative.

India ink capsule stain of

Cryptococcus neoformans

Methylene blue stain

of Corynebacterium

Acid-fast stain Red cells are acid-fast.

Blue cells are non-acid-fast.

Flagellar stain of Proteus vulgaris.

A basic stain was used to coat the flagella.

Endospore stain of Bacillus subtilis, showing endospores

(red) and vegetative cells (blue)