Methods for General and Molecular Microbiology

1055 Pages·2007·123.78 MB·English

by  Reddy

Methods
for
General
and
Molecular
Microbiology
3rd Edition
C. A. Reddy, Editor in Chief
T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt,
and L. R. Snyder, Editors
ASM
PRESS
Washington, D.C.
Cover images:
From Beniac, D. R., G. J. Czamota, B. L. Rutherford, E . 'F Ottensmeyer, and G. Harauz. 1997.
J. Microsc. 188:24-35; see chapter 4 (Beveridge et al.)
Rumen community, courtesy Frank Dazzo
G. L. Barron, University of Guelph; see chapter 42 (Thorn et al.)
M. McFall-Ngai, UW-Madison; see chapter 16 (Ciche et al.)
Copyright 0 2007 ASM Press
American Society for Microbiology
1752 N Street, N.W.
Washington, DC 20036-2904
Library of Congress Cataloginpin-Publication Data
Methods for general and molecular microbiology / C. A. Reddy, editor in chief ;
...
T.J. Beveridge [et al.], editors.-3rd ed.
D. : cm.
Includes indexes.
Rev. ed of: Methods for general and molecular bacteriology / Philipp Gerhardt, editor-in-chief ;
R.G.E. Murrav. Willis A. Wood. Noel R. Kriee. leditorsl. c1994.
-.
ISBN 978-1-55581-223-2 ~
1. Bacteriology-Laboratory manuals. 2. Microbiology-Laboratory manuals. I. Reddy, C. A.
11. Methods for general and molecular bacteriology.
[DNLM: 1. Microbiological Techniques. 2. Molecular Biology-methods. QW 25 M59 20071
QR65.M26 2007
616.9'2010786~22
2007018796
All Righhts Reserved
Printed in the United States of America
1 0 9 8 7 6 5 4 3 2 1
Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904,
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PREFACE
Methods in General and Molecular Microbiology (MGMM) is MGMB (Bacteriology) to MGMM (Microbiology).
a substantially revised, updated, and expanded version of MGMM is intended as a laboratory manual of methods to
its successful predecessor, Methods in General and Molecular complement traditional microbiology textbooks and sys-
Bacteriology (MGMB), published by ASM Press in 1994, tematic treatises of general microbiology. However, aside
with my colleague Philipp Gerhardt serving as the Editor- from a chapter on bacteriophages, this manual does not
in-Chief. The objective of MGMM is to provide a compre- cover viruses, algae, or protozoa.
hensive yet moderately priced book that will serve as a first MGMM is organized into sections corresponding to key
source for traditional methods of microbiology as well as subject areas of general bacterial and archaeal biology (mi-
modern molecular biological methods commonly used with croscopy, growth, metabolism, and molecular genetics), in-
microbial cells. Both previous editions of this manual, cluding a new section, Community and Genomic Analysis.
MGMB and Methodsfor General Bacteriology (MGB, 1991), This is followed by the new section on mycology and ap-
were popular not only in North America, but also in many pendices on laboratory safety and culture preservation.
other developed and developing countries worldwide. It is Each section is divided into chapters, and each chapter has
hoped that the audience for this edition will be even wider a table of contents to help the reader see the organization
and will include not only “card-carrying” microbiologists, of the chapter and easily locate a specific topic of interest.
but also scientists in allied disciplines working with mi- A decimal numbering system is used throughout the man-
crobes as experimental models or as tools for various ual to facilitate quick identification, cross referencing, and
biotechnological applications. It is hoped that this manual indexing. A comprehensive list of references is provided at
will be on the bookshelf of every serious practitioner of mi- the end of each chapter.
crobiology in academic, industrial, governmental, and clin- The editors refrained from imposing a single, rigid for-
ical laboratories and that it will serve as a rich resource of mat on the authors other than requesting them to use
methods for the seasoned professional as well as for under- MGMB as a guide in preparing their manuscripts. The ed-
graduate and graduate students and postdoctorals. itors also did not delineate in detail the topic assigned to an
The primary stimulus for launching this new edition of individual author(s). In spite of this, the chapters turned
the manual (MGMM) came from the fact that over a out to be remarkably consistent in their format and scope.
decade had elapsed since publication of the previous edi- For many of the chapter topics covered in this manual, the
tion (MGMB) and during this time not only new methods state of the art has so developed that entire books dedicated
but also new areas of microbiology (such as community and to the topic covered by a single chapter are currently avail-
genomic analysis) had emerged. Considering the fact that able. Therefore, in many instances, the chapter authors
MGMB and its predecessors had been used widely around have primarily described reliable methods that are widely
the globe, it was also felt that a new section on general applicable to basic studies on the topic covered. Each chap-
methods in mycology would make this edition even more ter presents sufficient background principles to understand
useful to its worldwide readership. Owing to this increased the how and why of a given method, followed by a step-by-
scope and the need to accommodate additional method- step description of the procedure. Common problems, pre-
ologies, this edition contains 47 total chapters (as com- cautions, and pitfalls of the methods are presented as a p
pared to 31 chapters in MGMB). The section on systemat- propriate. In many cases, commercial sources for
ics from the previous edition has been dropped: much of equipment and materials are given. These suggestions,
the still-relevant material in that section has been incor- however, are not meant either to endorse or to exclude a
porated into other sections, whereas outdated material particular product. Many of the commercial sources for
from that original section was omitted altogether. products are also readily ascertained from catalogues pub-
Inasmuch as this edition covers methods for microbes rep- lished by various commercial firms, from annual buyer’s
resenting all the three domains of life, i.e., Bacteria, guides of various journals, and from extensive information
Archaea, and Eukarya, the title has been broadened from available on the Internet.
xvii
xviii rn PREFACE
As mentioned above, two sections appear in this edition Transmission Electron Micrographs,” and “Atomic Force
for the first time. Rapid advances in genomics and genome- Microscopy” in the section Morphology and Ultrastructure;
based approaches have warranted the creation of the new “Energetics, Stoichiometry, and Kinetics of Microbial
section, Community and Genomic Analysis. The ability to Growth” and “General Methods To Investigate Microbial
determine complete genome sequences and metagenomes Symbioses” in the Growth section; “Bacterial Respiration,”
from microbial communities has revolutionized this field and “Carbohydrate Fermentations,” “Metabolism of Aromatic
has extended our ability to obtain valuable information on Compounds,” and two chapters on plant polymer-degrading
microbes that are yet to be cultured. Our rapidly expanding enzymes (“Cellulases, Hemicellulases, and Pectinases” and
knowledge in this area has been put together in seven excel- “Lignin and Lignin-Modifying Enzymes”) in the section on
lent chapters authored by leading researchers in this area. Metabolism; and “Measuring Spontaneous Mutation Rates,”
The new section on Mycology includes a comprehensive “Genetics of Archaea,” and “Genetic Manipulations Using
chapter on general methods and three excellent chapters on Phages” in the section on Molecular Genetics.
filamentous fungi, focusing on their physiology, metabolism, It goes without saying that a project of this size could
and genetic methods and on the principles and practice of not have been successfully completed without the excel-
DNA microarray technology. Again, each chapter is writ- lent cooperation and enormous effort on the part of the
ten by authorities in their respective areas of specialization. section editors and especially on the part of the authors,
In each of the sections retained from MGMB, existing many of whom have revised their chapters more than once;
chapters have been revised (quite extensively in many cases) the external referees, who provided rigorous and construc-
and a number of new chapters have been added. New au- tive critiques; and the dedicated publications staff of the
thors, revising existing chapters from the previous edition, ASM Press. Any corrections and constructive suggestions
have provided a fresh perspective, but in most cases the orig- for improvement from the users of this new edition are
inal authors of the analogous former chapter are credited. welcome.
Notable new chapters in MGMM, added to sections from
the previous edition, include “Laser Scanning Microscopy,” C. A. REDDY
“Computational Image Analysis and Reconstruction from Editor-in-Chief
CONTENTS
Reviewers / xi 8. Antigen-Antibody Reactions / 138
Contributors / xiii LUCY M.M UTHARIAAN D JOSEPH s. LAM
Preface / xvii
SECTION II
Acknowledgments / xix
GROWTH / 169
SECTION I
MORPHOLOGY Introduction / 171
AND ULTRASTRUCTURE / 1 JOHN A. BREZNAEKD,I TOR
Introduction to Morphology 9. Growth Measurement / 172
and Ultrastructure / 3 ARTHURL. KOCH
T. 1. BEVERIDGEDEI,T OR
10. Nutrition and Media / 200
1. Light Microscopy / 5 DAVIDE MERSONA ND JANE TANG
R. G. E. MURRAAYN D CARL E ROBINOW
11. Enrichment and Isolation / 21 5
2. Sampling and Staining for light ANDREATSE SKEH, ERIBERCTY PIONKA, JOHN G. HOLT,
Microscopy / 19 AND NOELR . KRIEG
TERRJY. B EVERIDGJOEH, N R. LAWRENCE,
12. Culture Techniques / 270
AND ROBERTG . E. MURRAY
SYEDA . HASHSHAM
3. Laser Scanning Microscopy / 34
13. Energetics, Stoichiometry, and Kinetics
J. R. LAWRENCANED T. R. NEU
of Microbial Growth / 286
SYEDA . HASHSHAAMN D SAMW . BAUSHKE
4. Electron Microscopy / 54
TERRJY. B EVERIDGED, IANNME OYLES,
14. Physicochemical Factors
AND BOBH ARRIS in Growth / 309
JOHN A. BREZNAAKN D RALPHN . COSTILOW
5. Computational Image Analysis
and Reconstruction from Transmission
15. Phenotypic Characterization
Electron Micrographs / 82
and the Principles of Comparative
GEORGHE ARAUZ
Systematics / 330
BRIANI .T INDALJLO,H ANNES SIKORSKI,
6. Atomic Force Microscopy / 96 ROBERTA . SMIBERT, AND NOELR . KRIEG
YVES F. DUFRBNE
16. General Methods To Investigate
7. Cell Fractionation / 108 Microbial Symbioses / 394
SUSAFN. K OVALA ND G. DENNISSP ROTT TODDA . ClCHE AND SHANA K. GOFFREDI
vii
viii w CONTENTS
SECTION Ill 28. Measuring Spontaneous Mutation
METABOLISM / 421 Rates / 676
PATRICILA. FOSTER
Introduction to Metabolism / 423
C. A. REDDYE,D ITOR 29. Transposon Mutagenesis / 684
SILVIA ROSSBACAHN D FRANSJ. DE BRUIJN
17. Physical Analysis and Purification
Methods / 424 30. Plasmids / 709
SCOT B. MULROONEWYI,L LISA . WOOD, MARCELEO. TOLMASLKUYIS, A . ACTIS,
AND J. R. PATEREK TIMOTHJ. YW ELCH, AND JORGE H. CROSA
18. Chemical Analysis / 462 31. Gene Transfer in Gram-Negative
LACYD ANIELRS,I CHARSD. HANSON, Bacteria / 735
AND JANE A. PHILLIPS JOSEPH E. PETERS
19. Enzymatic Activity / 504
32. Genetic Exchange in Gram-Positive
ROBERPT. HAUSINGEARND ALLENT. PHILLIPS Bacteria / 756
CHRISTOPJH. EKRRI STICH,C HRISTINEE. S ALOMON,
20. Permeability and Transport / 527
AND GARYM . DUNNY
ROBERET. MARQUIS
33. Genetics of Archaea / 800
21. Bacterial Respiration / 539
KEVINR . SOWERSPA, ULH . BLUM,
ROBERPT. GUNSALUGS,A RYC ECCHINI,
AND SHILADITYDAA SSARMA
AND IMKES CHRODER
34. Genetic Manipulations Using
22. Carbohydrate Fermentations / 558
Phages / 825
R. MEGANATHAYNAM, INIR ANGANATHAN,
c. GRAHAME HATFULDL,E BORAJHA COBS-SERA,
AND A. REDDY
MICHELLHE. LARSENA,N D WILLIAMR. JACOBS, JR.
23. Metabolism of Aromatic
Compounds / 586 SECTION V
JEROME J. KUKOR, BORISW AWRIK, COMMUNITY AND GENOMIC
AND GERBEJN. ZYLSTRA ANALYSIS / 839
24. CelI u lases, Hemic ellu lases, Introduction to Community
and Pectinases / 596 and Genomic Analysis / 841
MICHAEEL. HIMMEJLO,H N 0. BAKER, THOMAMS. SCHMIDETD, ITOR
WILLIAMS. ADNEYA,N D STEPHERN. DECKER
35. Characterization of Bacterial Genome
25. Lignin and Lignin-Modifying Sequences by Similarity
Enzymes / 611
Searching / 842
CARLOGS . DOSORETAZN D C. A. REDDY WILLIAMR. PEARSON
SECTION IV
MOLECULAR GENETICS / 621 36. Reconstructing and Interpreting
Evolutionary Relationships / 856
Introduction to Molecular CHRISTOPHE J. DOUADAYN D CAMILLA L. NESB0
Genetics / 623
C. A. REDDYE,D ITOR 37. Microbial Nucleotide Fingerprints
L. R. SNYDERC,O -EDITOR in Nature / 869
DAVIDM .K ARL
26. Similarity Analysis of DNAs / 624
JOHN L. JOHNSON AND WILLIAM B. WHITMAN 38. Construction of BAC and Fosmid
Libraries from Naturally Occurring
27. Nucleic Acid Analysis / 653 Microbial Populations / 879
WILLIAM HENDRICKSAONND DONW ALTHERS EDWARED DELONG
Contents w ix
Single Cell Identification by Fluorescence Physiology, Metabolism, and Molecular
In Situ Hybridization / 886 Aspects of Filamentous Fungi / 952
BERNHARMD. FUCHSJ, AKOBPE RNTHALER, GEORGEA . MARZLUF
AND RUDOLAF MANN
Microbiological and Genetic Methods
Measurement of rRNA Abundance for Filamentous Fungi / 965
by Hybridization with ROWLANHD. DAVIS
Oligodeoxynucleotide Probes / 897 AND A. JOHNC LUTTERBUCK
DANIELH . BUCKLEAYN D THOMASM . SCHMIDT
Principles and Practice of DNA
Analysis of Microbial Communities Microarray Technology / 978
with Denaturing Gradient Gel KRISHNAMURTNHAYT ARAJAN,
Electrophoresis and Terminal MATTHEWJ. MARTON,
Restriction Fragment Length AND ALANG . HINNEBUSCH
Polymorphism / 909
TERENCLE. MARSHA ND CINDYH . NAKATSU APPENDICES
SECTION VI 46. Laboratory Safety / 997
MYCOLOGY / 925 W. EMMETBT ARKLEAYN D CLAUDIAA. MICKELSON
Introduction to Mycology / 927 47. Culture Preservation / 1019
GEORGAE . MARZLUEF,D ITOR ROBERTL . GHERNAAN D C. A. REDDY
42. Methods for Studying Terrestrial Fungal Author lndex / 1035
Ecology and Diversity / 929
R. G. THORNJ,. SCOTT, AND M. A. LACHANCE Subject lndex / 1039
MORPHOLOGY AND
UL TRASTRU CTU RE
Introduction to Morphology and 5. Computational Image Analysis and
UI t rastr uctur e 3 Reconstruction from Transmission
T. J. BEVERIDGE, Editor Electron Micrographs 82
GEORGE HARAUZ
1. Light Microscopy 5
R. G. E. MURRAY AND CARL F. ROBINOW 6. Atomic Force Microscopy 96
YVES F. DUFRENE
2. Sampling and Staining for Light
Microscopy 19 7. Cell Fractionation 108
TERRY J. BEVERIDGE, JOHN R. LAWRENCE, SUSAN E KOVAL AND G. DENNIS SPROTT
AND ROBERT G. E. MURRAY
8. Antigen-Antibody Reactions 138
3. Laser Scanning Microscopy 34 LUCY M. MUTHARIA AND JOSEPH S. LAM
J. R. LAWRENCE AND T. R. NEU
4. Electron Microscopy 54
TERRY J. BEVERIDGE, DIANNE MOYLES,
AND BOB HARRIS
Introduction to Morphology and Ultrastructure
T. J. BEVERIDGE
Microbiology and microscopy have enjoyed a strong syner- macromolecules within cellular space so that their location
gistic union since the earliest times of scientific investiga- can be seen (e.g., within nucleoids, division septa, secretory
tion. It is impossible for us to understand, today, how the apparatuses, endospores, etc.). Confocal laser scanning
crude lenses of Antonie van Leeuwenhoek or Robert microscopes (CLSMs) remain relatively expensive instru-
Hooke could have achieved the resolution they did, or how ments (compared to more simple light microscopes), but
the almost happenstance discovery of a fundamental differ- they are now readily available at the university, college, and
ential stain by Christian Gram would eventually partition (even) departmental levels. Because of their expense and
bacteria into two fundamental categories, gram-positive technical sophistication, most CSLMs are operated by a
and gram-negative bacteria. Because the individual cells of dedicated technical staff member who will aid students and
prokaryotes are so minute, they cannot be seen without researchers alike. Much of our current knowledge of the
some sort of microscopy. Accordingly, there necessarily has growth and spatial distribution of cells within biofilms has
been a fine history between microscopy and microbiology. come from the use of CSLMs.
Even the best light microscopes that are available today It is safe to say that almost all of our knowledge of the
can discriminate only shape and form unless distinctive fine structures in prokaryotes has been derived by using
staining procedures are used for differentiation of fine electron microscopes. Scanning electron microscopes
structures and added contrast. Consequently, other kinds of (SEMs) have clarified the topography of biofilms, colonies,
microscopy, often using wavelengths and energies different and (sometimes) individual cells. So-called environmental
from those of light, have been resorted to. Yet, light mi- SEMs can give images of specimens while they are partially
croscopy continues to be a hallmark of microbiology, and hydrated under high humidity. Transmission electron mi-
the first two chapters of this section cover the general croscopes (TEMs), though, have been our most important
methods employed. Chapter 1 outlines the basic compo- tool in microbiology for determining the macromolecular
nents of a simple compound light microscope and their op- structures of prokaryotes. Cell surfaces, juxtapositions of
eration. Chapter 2 broaches the various manipulations to enveloping layers, distributions of cytoplasmic constit-
microorganisms before they can be viewed; these include uents, intramembranous inclusions, and cytoplasmic parti-
the simple isolation of cells, chemical fixation, and both cles (e.g., polyphosphate, sulfur, and polyhydroxyalkoanate
general and specific staining protocols for cells and con- granules) have all been seen at high resolution, sometimes
stituent structures. confirming less resolving pre-TEM light microscopic evi-
It cannot be emphasized too much how important are dence.
these tried and true optical techniques to microbiology. It Electron microscopy requires difficult and (often) ex-
saddens those of us who specialize in microscopic imaging tensive preprocessing before specimens can be viewed.
to sometimes see how neglected and infrequently used is Frequently these procedures can induce artifacts that are
light microscopy in modern microbiological research labo- difficult to recognize for the uninitiated researcher. For this
ratories. Some laboratories do not even possess a workable reason, chapter 4 outlines tried and true protocols, using
light microscope! Researchers in these laboratories seem more traditional avenues for TEM, which most microbiol-
confident in their ability to maneuver relatively small mol- ogy laboratories should be capable of. Here, for even the
ecules of DNA as they manipulate the genetic message in difficult procedure of thin sectioning, cells can be grown,
cells and have little desire to actually visualize the cellular chemically fixed, stained, and embedded in plastic without
results. In these laboratories, possible altered phenotypes, specialized equipment. These samples can then be taken to
microbial contaminants, and lethal or growth effects are in-house TEM facilities or shipped over larger distances.
rarely monitored by light microscopy during such refined Because SEMs and TEMs are expensive to buy, operate,
molecular biological manipulations. Even more, by mi- and maintain, these microscopes are usually clustered
croscopy’s absence, a clear tangible distance is established within a university facility containing ancillary equipment
between the experimenter and that being experimented for the manipulation of your samples. Specimens can be
on, which lends a certain remoteness to the experimental thin sectioned, TEM grids can be prepared, and an opera-
process. We need to rediscover our traditional microscopic tor can be supplied for viewing specimens. Remember, the
roots in microbiology, and chapters 1 and 2 are a required technical staff members within these electron microscopy
knowledge base for students and researchers alike. facilities are expert microscopists but they may not be ex-
Chapter 3 covers confocal laser scanning microscopy, perienced in microbiology. For this reason, it can be ad-
which is fast becoming an essential part of microbiology’s vantageous to network with an experienced microbiology
imaging armament. Good light microscopes combined with structuralist. Over the last decade, a renaissance in micro-
excellent coherent light sources (lasers) provide accurate biological ultrastructure has gradually occurred with the
resolution plus defined focal sections through relatively advent of cryogenic techniques. Here, cells are vitrified to
thick specimens. Specialized (but often user-friendly) com- preserve their native structures by ultrarapid freezing. One
puter software allows the easy manipulation of serial focal such cryogenic method, freeze substitution, is described in
sections so that cells located in x, y, and 2 axial planes of chapter 4 since it has brought remarkable clarity to a num-
the sample can be visualized and stacked to form three- ber of microbial structures, especially the enveloping layers
dimensional images. Fluorescent probes, excited at specific of bacteria. With some effort and dedication, freeze substi-
wavelengths, provide exquisite regional discrimination of tution can be done in a typical microbiology laboratory.
3
4 MORPHOLOGY AND ULTRASTRUCTURE
However, more dedicated cryogenic techniques, such as broken or permeabilized so that their cellular constituents
those required for the production of frozen foils or frozen can be fractionated to purity. It also gives valuable advice
hydrated thin sections, need more dedicated technical skill on how to determine so-called biochemical signatures of
and equipment (including cryoTEMs with electron energy structural components (e.g., the lipopolysaccharide of the
filters) and are best left to the experts. gram-negative outer membrane), which is essential for
As previously mentioned, user-friendly computer soft- tracking the various fractionations and (eventually) deter-
ware is an essential part of CSLMs and is included with mining purity. Many of the described methods are used to
them. However, computer manipulation of electron micro- obtain the components of cells that are eventually imaged
graphs is more difficult and uses sophisticated Fourier trans- by the TEM techniques described in chapter 4.
formation- or correlation averaging-based analysis to reduce One of the more difficult aspects of any form of mi-
the signal-to-noise ratio within the raw images. Chapter 5 croscopy is to be able to unequivocally identify specific
is a short but comprehensive explanation of image en- structures or macromolecules within a cell. Here, a specific
hancement in electron microscopy. It also includes the ex- probe to label such a constituent is an invaluable tool. One
citing new techniques of electron spectroscopic imaging (an of the best probes is an antibody specific to the structure of
electron-filtering method) and tomography (three-dimen- interest. Chapter 8 describes the techniques used to obtain
sional imaging by tilting the specimen) as applied to such such probes and how to ensure their specificity. These
small particles as ribosomal subunits so that rRNA can be probes can be polyvalent antibodies obtained from an ani-
seen as it folds its way around putative ribosomal proteins. mal after immunization or monoclonal antibodies devel-
Scanning probe microscopy, (SPM) which is represented oped through myeloma-spleen hybridomas. Highest speci-
by atomic force microscopy (AFM) in chapter 6, is an en- ficity is via monoclonal antibodies with discrimination of a
tirely different type of microscopy in that no lenses are used. single epitope, but highest labeling capacity is through poly-
Here, a sharp tip on a bendable cantilever is dragged over a clonal antibodies capable of labeling many epitopes at a sin-
sample in a raster pattern with the tip forced up and down gle time. Both factors have to be weighed against one an-
depending on the contours found on the specimen. The tip other when determining the best probe for your sample.
actually is in contact with the specimen and, depending on Once the immunological probe has been obtained, it must
the physical attributes of the sample’s surface, can detect be labeled with a visual marker that can be detected by mi-
the topography of macromolecules and even atoms. Unlike croscopy. For light microscopy, this label is usually a fluo-
other forms of scanning probe microscopy, such as scanning rescent marker (e.g., fluorescein; see chapters 2 and 3),b ut
tunneling microscopy, AFM can be done under water, for electron microscopy an electron-dense marker (e.g., col-
which makes it a valuable microscopy for biological sub- loidal gold; see chapter 4) is needed. For AFM, the probe
stances. Chapter 6 explains the basic setup of the atomic must be large enough to be visualized against the typical to-
force microscope and how AFM is making inroads in mi- pography of the cell (e.g., a bacteriophage that is specific for
crobiology. Another valuable trait of the microscope is that the surface).
the tip, on a cantilever of a known spring constant, can Together, the chapters in this section are an integration
probe the physical nature of the sample to determine such of the methods generally used in microbiology to visualize
important traits as elasticity, viscosity, and adhesion. At one microorganisms and their constituent parts, along with the
time atomic force microscopes were highly specialized and techniques required for component isolation and visual de-
infrequently encountered in a research setting, but now tection. Although these chapters make a correlated union,
they are an item that all research-intensive universities they will also be helpful when applied to some of the tech-
have. niques described in other sections and chapters of this book.
It is one thing to discern the structures of microorgan- Indeed, the images that have been chosen to portray the
isms by looking at intact cells, but it is an entirely different various stains, cells, and structures in this section have also
and more demanding task to decipher the contours of their been chosen so as to help illustrate the microorganisms
individual components. For this, the components usually mentioned in other chapters of the book. One of the great
have to be isolated and purified. Once these steps are ac- pleasures of combining microbiology and microscopy is to
complished, these cellular constituents can not only be vi- enjoy the simplicity and beauty of microbial shape and
sualized by microscopy (usually by using either TEM or form, but each image also possesses a wealth of scientific in-
AFM), they can also be used for biochemical analyses to de- formation. This marriage of science and visual enjoyment
termine their chemical makeups and possible activities. has its roots in the 20th century and continues today with
Chapter 7 describes the manner in which microbes can be more technical sophistication and interpretative skills.