Ebook Histology text and atlas (6th edition): Part 1

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Sixth Edition

HISTOLOGY
A Text and Atlas
with Correlated Cell
and Molecular Biology

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Michael H. Ross (1930–2009)

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Sixth Edition

HISTOLOGY
A Text and Atlas
with Correlated Cell
and Molecular Biology

Michael H. Ross, PhD (deceased)

Professor and Chairman Emeritus
Department of Anatomy and Cell Biology
University of Florida College of Medicine
Gainesville, Florida

Wojciech Pawlina, MD
Professor and Chair
Department of Anatomy
Department of Obstetrics and Gynecology
Assistant Dean for Curriculum Development and Innovation
Mayo Medical School
College of Medicine, Mayo Clinic
Rochester, Minnesota

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Acquisitions Editor: Crystal Taylor
Product Manager: Jennifer Verbiar
Designer: Doug Smock
Compositor: MPS Limited, A Macmillan Company
Sixth Edition
Copyright © 2011 <<2006, 2003, 1995, 1989, 1985>> Lippincott Williams & Wilkins, a Wolters Kluwer business.

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All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission
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Library of Congress Cataloging-in-Publication Data
Ross, Michael H.
Histology: a text and atlas: with correlated cell and molecular biology/Michael H. Ross, Wojciech Pawlina.—6th ed.
p. ; cm
Includes bibliographical references and index.
ISBN 978-0-7817-7200-6 (alk. paper)
1. Histology. 2. Histology—Atlases. I. Pawlina, Wojciech. II. Title.
[DNLM: 1. Histology—Atlases. QS 517 R825h 2011]
QM551.R67 2011
611’.018—dc22
2010024700
DISCLAIMER
Care has been taken to confirm the accuracy of the information present and to describe generally accepted practices. However, the authors, editors,
and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this information
in a particular situation remains the professional responsibility of the practitioner; the clinical treatments described and recommended may not be
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72006_FM 15/07/10 6:59 PM Page v

This edition is dedicated to my wife Teresa Pawlina whose love, patience, and endurance created
safe havens for working on this project and to my children Conrad Pawlina and Stephanie Pawlina
whose stimulation and excitement have always kept my catecholamine levels high.

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Preface
This sixth edition of Histology: A Text and Atlas with Correlated
Cell and Molecular Biology continues a tradition of providing
medical, dental, and allied health science students with a textual and visual introduction to histology with correlative cell
biology. As in previous editions, this book is a combination
“text-atlas” in that standard textbook descriptions of histologic
principles are supplemented by illustrations and photographs.
In addition, separate atlas sections follow each chapter and provide large-format, labeled atlas plates with detailed legends
highlighting elements of microanatomy. Histology: A Text and
Atlas is therefore “two books in one.”
Significant modifications have been made in this edition
in order to create an even more useful and understandable approach to the material:
Updated cellular and molecular biology. Material introduced in the fifth edition has been updated to include the latest advancements in cellular and molecular biology. The sixth

edition focuses on selected information to help students with
overall comprehension of the subject matter. To accommodate reviewers’ suggestions, the sixth edition also integrates
new cell biology information into several chapters. For instance, the cell biology of endothelial cells has been added to
the discussion of the cardiovascular system; a section on primary cilia, including their structure and function, was added
to the epithelial tissue chapter; a new clinical nomenclature
for cells involved in hemopoiesis and a detailed description of
the respiratory burst reaction in neutrophils were added to
the chapter on blood; new information and diagrams of nerve
fiber regeneration were added to the nerve tissue chapter; and
the cell biology of taste receptors was incorporated into the
chapter on the digestive system.
Reader-friendly innovations. The book has been redesigned
in an attempt to provide more ready access to important concepts and essential information. Additional color font is used
in the body of the text. Important concepts are listed as sentence headings. Features of cells, tissues, and organs and their
functions, locations, and other relevant short phrases are formatted as bulleted lists that are clearly identifiable in the body
of the text by oversized color bullets. Essential terms within each

specific section are introduced in the text in an eye-catching
oversized red bolded font that clearly stands out from the remaining black text. Text containing clinical information or the
latest research findings is presented in blue, with terminology
pertaining to diseases, conditions, symptoms, or causative
mechanisms in oversized bolded blue. The clinical sections of
the text are easily found within each chapter.
Emphasis on features. Many of the pedagogic features

from the last edition have been refined, and some new features have been added:
• More summary tables are included to aid students in learning and reviewing material without having to rely on
strict memorization of data. These include a review table of
the specializations in the apical domains of epithelial cells
and a table of features of adipose tissue. Many tables have

been updated and modified.
• Previous clinical and functional correlations boxes have
been replaced with Clinical Correlation and Functional
Consideration Folders. More new folders have been added
to each chapter, and existing folders have been redesigned,
updated, enhanced, and illustrated with new diagrams and
images of clinical specimens. New folders contain clinical
information related to the symptoms, photomicrographs
of diseased tissues or organs, short histopathological
descriptions, and treatment of specific diseases. Important
terms have been highlighted with oversized bolded text.
While the information in these folders might be
considered ancillary material, it demonstrates the
functional impact and clinical significance of histology.
• More Atlas Plates have been added to the atlas section at
the end of each chapter. Several orientation micrographs
were added to the summary box in the atlas section. Atlas
plates for the blood chapters have been completely redesigned so as to show both mature forms of blood cells
and the stages through which they pass during
hemopoiesis. Many plates have been replaced with vibrant
digital images.
• More new figures and illustrations have also been added,
and about one-third of all old figures have been redrawn for

vii

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greater clarity and conceptual focus. This sixth edition incorporates many new clinical images and photomicrographs

to illustrate information in the clinical correlation folders.
Many new high-resolution digital photomicrographs have
been integrated into each chapter.
• New design. A bright, energetic text design sets off the new
illustrations and photos and makes navigation of the text
even easier than in previous editions.

As in the last five editions, all of the changes were undertaken with student needs in mind; namely, to understand the
subject matter, to become familiar with the latest information, and to be able to practically apply newfound knowledge.
Wojciech Pawlina

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Acknowledgments
This sixth edition of Histology: A Text and Atlas with Correlated Cell and Molecular Biology reflects continued improvement on previous editions. The changes that have been made
come largely from comments and suggestions by students
who have taken the time and effort to tell us what they like
about the book and, more importantly, how it might be improved to help them better understand the subject matter.
The majority of such comments and suggestions have been
incorporated into this new edition.
Many of our colleagues who teach histology and cell biology courses were likewise most helpful in creating this new
edition. Many of them suggested a stronger emphasis on clinical relevance, which we responded to as best we could within
page limitations. Others were most helpful in providing new
micrographs, suggesting new tables, and redrawing existing
diagrams and figures.
Specifically, we owe our thanks to the following reviewers,
both students and faculty, who spent considerable time and
effort to provide us with corrections and suggestions for improvement. Their comments were a valuable source of information in planning this sixth edition.

Craig A. Canby, PhD
Des Moines University
Des Moines, Iowa

Irwin Beitch, PhD
Quinnipiac University
Hamden, Connecticut

Jolanta Durski, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Paul B. Bell, Jr., PhD
University of Oklahoma
Norman, Oklahoma

William D. Edwards, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

David E. Birk, PhD
University of South Florida, College of Medicine
Tampa, Florida

Bruce E. Felgenhauer, PhD
University of Louisiana at Lafayette
Lafayette, Louisiana

Christy Bridges, PhD
Mercer University School of Medicine

Macon, Georgia

Amos Gona, PhD
University of Medicine & Dentistry of New Jersey
Newark, New Jersey

Benjamin S. Bryner, MD
University of Michigan Medical School
Ann Arbor, Michigan

Ervin M. Gore, PhD
Middle Tennessee State University
Murfreesboro, Tennessee

Stephen W. Carmichael, PhD
College of Medicine, Mayo Clinic
Rochester, Minnesota
John Clancy, Jr., PhD
Loyola University Medical Center
Maywood, Illinois
Rita Colella, PhD
University of Louisville School of Medicine
Louisville, Kentucky
Iris M. Cook, PhD
State University of New York Westchester Community College
Valhalla, New York

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Joseph P. Grande, MD, PhD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Gavin R. Lawson, PhD
Western University of Health Sciences
Bridgewater, Virginia

Joseph A. Grasso, PhD
University of Connecticut Health Center
Farmington, Connecticut

Susan LeDoux, PhD
University of South Alabama
Mobile, Alabama

Jeremy K. Gregory, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Karen Leong, MD
Drexel University College of Medicine
Philadelphia, Pennsylvania

Brian H. Hallas, PhD
New York Institute of Technology
Old Westbury, New York

A. Malia Lewis, PhD
Loma Linda University
Loma Linda, California

Charlene Hoegler, PhD
Pace University
Pleasantville, New York

Wilma L. Lingle, PhD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Cynthia J. M. Kane, PhD
University of Arkansas for Medical Sciences
Little Rock, Arkansas

Frank Liuzzi, PhD
Lake Erie College of Osteopathic Medicine
Bradenton, Florida

Thomas S. King, PhD
University of Texas Health Science Center at San Antonio
San Antonio, Texas

Donald J. Lowrie, Jr., PhD
University of Cincinnati College of Medicine
Cincinnati, Ohio

Penprapa S. Klinkhachorn, PhD
West Virginia University

Morgantown, West Virginia

Andrew T. Mariassy, PhD
Nova Southeastern University College of Medical Sciences
Fort Lauderdale, Florida

Bruce M. Koeppen, MD, PhD
University of Connecticut Health Center
Farmington, Connecticut

Geoffrey W. McAuliffe, PhD
Robert Wood Johnson Medical School
Piscataway, New Jersey

Beverley Kramer, PhD
University of the Witwatersrand
Johannesburg, South Africa

Kevin J. McCarthy, PhD
Louisiana State University Health Sciences Center
Shreveport, Louisiana

Craig Kuehn, PhD
Western University of Health Sciences
Pomona, California

David L. McWhorter, PhD
Philadelphia College of Osteopathic Medicine—
Georgia Campus
Suwanee, Georgia

Nirusha Lachman, PhD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Joseph J. Maleszewski, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Priti S. Lacy, PhD
Des Moines University, College of Osteopathic Medicine
Des Moines, Iowa

Fabiola Medeiros, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

H. Wayne Lambert, PhD
West Virginia University
Morgantown, West Virginia

William D. Meek, PhD
Oklahoma State University, College of Osteopathic Medicine
Tulsa, Oklahoma

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Karuna Munjal, MD
Baylor College of Medicine
Houston, Texas

Mary Rheuben, PhD
Michigan State University
East Lansing, Michigan

Lily J. Ning, MD
University of Medicine & Dentistry of New Jersey, New Jersey
Medical School
Newark, New Jersey

Jeffrey L. Salisbury, PhD
College of Medicine, Mayo Clinic
Rochester, Minnesota

Diego F. Nino, PhD
Louisiana State University Health Sciences Center, Delgado
Community College
New Orleans, Louisiana
Sasha N. Noe, DO, PhD
Saint Leo University
Saint Leo, Florida
Joanne Orth, PhD
Temple University School of Medicine
Downingtown, Pennsylvania
Nalini Pather, PhD
University of New South Wales
Sidney, Australia

Tom P. Phillips, PhD
University of Missouri
Columbia, Missouri
Stephen R. Planck, PhD
Oregon Health and Science University
Portland, Oregon
Dennifield W. Player, BS
University of Florida
Gainesville, Florida
Harry H. Plymale, PhD
San Diego State University
San Diego, California
Rebecca L. Pratt, PhD
West Virginia School of Osteopathic Medicine
Lewisburg, West Virginia

Young-Jin Son, PhD
Drexel University
Philadelphia, Pennsylvania
David K. Saunders, PhD
University of Northern Iowa
Cedar Falls, Iowa
John T. Soley, DVM, PhD
University of Pretoria
Pretoria, South Africa
Anca M. Stefan, MD
Touro University College of Medicine
Hackensack, New Jersey
Alvin Telser, PhD
Northwestern University Medical School

Chicago, Illinois
Barry Timms, PhD
Sanford School of Medicine, University of South Dakota
Vermillion, South Dakota
James J. Tomasek, PhD
University of Oklahoma Health Science Center
Oklahoma City, Oklahoma
John Matthew Velkey, PhD
University of Michigan
Ann Arbor, Michigan
Daniel W. Visscher, MD
University of Michigan Medical School
Ann Arbor, Michigan

Margaret Pratten, PhD
The University of Nottingham, Medical School
Nottingham, United Kingdom

Anne-Marie Williams, PhD
University of Tasmania, School of Medical Sciences
Hobart, Tasmania

Rongsun Pu, PhD
Kean University
East Brunswick, New Jersey

Joan W. Witkin, PhD
Columbia University, College of Physicians and Surgeons
New York, New York

Romano Regazzi, PhD
University of Lausanne, Faculty of Biology and Medicine
Lausanne, Switzerland

Alexandra P. Wolanskyj, MD
College of Medicine, Mayo Clinic
Rochester, Minnesota

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Robert W. Zajdel, PhD
State University of New York Upstate Medical University
Syracuse, New York
Renzo A. Zaldivar, MD
Aesthetic Facial & Ocular Plastic Surgery Center
Chapel Hill, North Carolina

A few colleagues have made especially notable contributions
to this textbook. We are extremely grateful to Dr. Renzo Zaldivar from the Aesthetic Facial & Ocular Plastic Surgery Center in Chapel Hill, North Carolina for providing us with
clinical images and content for several clinical correlations folders in the chapter on the eye. Our deep appreciation goes to
Drs. Fabiola Medeiros from Mayo Clinic and Donald Lowrie,
Jr., from the University of Cincinnati College of Medicine for
providing original glass slides of the highest quality of several
specimens. In addition, Todd Barnash from the University
of Florida provided invaluable technical assistance with the

xii

digitized text, figures, and photomicrographs. Thanks also go
to Denny Player for his superb technical assistance with
electron microscopy.
All of the new art in this edition was created by Rob Duckwall and his wife Caitlin Duckwall from the Dragonfly Media
Group (Baltimore, MD). Their expertise in creating innovative and aesthetically-pleasing artwork is greatly appreciated.
The authors also wish to extend special thanks to Jennifer
Verbiar, our managing editor, and her predecessor Kathleen
Scogna, who provided expertise during the majority of the development process. Our editors’ problem solving and technical skills were crucial to bringing this text to fruition, and their
contributions to the sixth edition were priceless. Our thanks
goes to Arijit Biswas, the Project Manager of MPS Limited,
A Macmillan Company in New Delhi, India, and his staff of
compositors for an excellent job in putting together this
complex and challenging publication. Finally, a special thanks
to Crystal Taylor for her support throughout the development
of the book. Her diligence is much appreciated.

72006_FM 15/07/10 7:00 PM Page xiii

Contents
Preface | vii
Acknowledgments | ix

1. METHODS | 1
Overview of Methods Used in Histology | 1
Tissue Preparation | 2
Histochemistry and Cytochemistry | 3
Microscopy | 13
Folder 1.1

Clinical Correlation: Abnormalities in
Microtubules and Filaments | 68

Folder 5.2

Clinical Correlation: Primary Ciliary
Dyskinesia | 120

Folder 2.3

Clinical Correlation: Abnormal Duplication
of Centrioles and Cancer | 72

Folder 5.3

Clinical Correlation: Junctional Complexes
as a Target of Pathogenic Agents | 128

Folder 5.4

Functional Considerations: Basement
Membrane and Basal Lamina
Terminology | 138

Folder 5.5

Functional Considerations: Mucus and
Serous Membranes | 150

3. THE CELL NUCLEUS | 75

Intramembranous Bone Formation | 252

9. ADIPOSE TISSUE | 254
Overview of Adipose Tissue | 254
White Adipose Tissue | 254
Brown Adipose Tissue | 259

Folder 6.1

Clinical Correlation: Collagenopathies | 170

Folder 6.2

Clinical Correlation: Sun Exposure and
Molecular Changes in Photoaged Skin | 173

Folder 6.3

Clinical Correlation: Role of Myofibroblasts in
Wound Repair | 183

Folder 9.1

Clinical Correlation: Obesity | 261

Folder 6.4

Functional Considerations: The Mononuclear
Phagocytotic System | 185

Folder 9.2

Clinical Correlation: Adipose Tissue Tumors | 262

Folder 9.3

Folder 6.5

Clinical Correlation: The Role of Mast Cells
and Basophils in Allergic Reactions | 188

Clinical Correlation: PET Scanning and
Brown Adipose Tissue Interference | 264

Atlas Plates
Plate 4

Loose and Dense Irregular Connective
Tissue | 192

Plate 5

Dense Regular Connective Tissue, Tendons, and
Ligaments | 194

Plate 6

Elastic Fibers and Elastic Lamellae | 196

7. CARTILAGE | 198

Clinical Correlation: Osteoarthritis | 199

Folder 10.3

Clinical Correlation: Hemoglobin Disorders | 276

Clinical Correlation: Malignant Tumors of
the Cartilage; Chondrosarcomas | 208

Folder 10.4

Atlas Plates
Plate 7

Clinical Correlation: Inherited Disorders of
Neutrophils; Chronic Granulomatous Disease
(CGD) | 281

Hyaline Cartilage | 210

Folder 10.5

Plate 8

Cartilage and the Developing Skeleton | 212

Clinical Correlation: Hemoglobin Breakdown
and Jaundice | 281

Plate 9

Folder 11.1

Folder 8.3

Clinical Correlation: Nutritional Factors
in Bone Formation | 234

Functional Considerations: Muscle Metabolism
and Ischemia | 316

Folder 11.2

Folder 8.4

Functional Considerations: Hormonal
Regulation of Bone Growth | 242

Clinical Correlation: Muscular Dystrophies—
Dystrophin and Dystrophin- Associated
Proteins | 319

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Folder 11.3

Functional Considerations: The Sliding
Filament Model | 323

Folder 11.4

Clinical Correlation: Myasthenia Gravis | 325

Folder 11.5

Functional Considerations: Comparison of
the Three Muscle Types | 337

Atlas Plates
Plate 21

Skeletal Muscle I | 340

Plate 22

Skeletal Muscle II and Electron Microscopy | 342

Plate 23

Myotendinal Junction | 344

Plate 24

Cardiac Muscle | 346

Plate 25

Cardiac Muscle, Purkinje Fibers | 348

14. LYMPHATIC SYSTEM | 440
Overview of the Lymphatic System | 440
Cells of the Lymphatic System | 441
Lymphatic Tissues and Organs | 453
Folder 14.1

Functional Considerations: Origin of the
Names T Lymphocyte and B Lymphocyte | 447

Folder 14.2

Clinical Correlation: Hypersensitivity
Reactions | 447

Folder 14.3

Clinical Correlation: Human Immunodeficiency
Virus (HIV) and Acquired Immunodeficiency
Syndrome (AIDS) | 455

Folder 14.4

Clinical Correlation: Reactive (Inflammatory)
Lymphadenitis | 466

Atlas Plates
Plate 36

Palatine Tonsil | 476

Folder 15.2

Functional Considerations: Skin Color | 499

Folder 15.3

Functional Considerations: Hair Growth
and Hair Characteristics | 504

Folder 15.4

Functional Considerations: The Role of
Sebum | 505

Folder 15.5

Clinical Correlation: Sweating and
Disease | 507

Folder 15.6

Clinical Correlation: Skin Repair | 512

Atlas Plates
Plate 42

Skin I | 514

Plate 43

of Taste | 533

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Folder 16.2

Clinical Correlation: Classification of
Permanent (Secondary) and Deciduous
(Primary) Dentition | 534

Folder 16.3

Clinical Correlation: Dental Caries | 547

Folder 16.4

Clinical Correlation: Salivary Gland Tumors | 555

Atlas Plates
Plate 48

Lip, A Mucocutaneous Junction | 556

Atlas Plates
Plate 65

Liver I | 656

Alveoli | 678
Blood Supply | 687
Lymphatic Vessels | 687
Nerves | 687

Folder 17.1

Clinical Correlation: Pernicious Anemia
and Peptic Ulcer Disease | 578

Folder 17.2

Clinical Correlation: Zollinger-Ellison
Syndrome | 580

Folder 17.3

Functional Considerations: The Gastrointestinal
Endocrine System | 581

Folder 17.4

Functional Considerations: Digestive and
Absorptive Functions of Enterocytes | 587

Atlas Plates
Plate 69

Olfactory Mucosa | 688

Folder 17.5

Functional Considerations: Immune Functions
of the Alimentary Canal | 595

Plate 70

Larynx | 690

Clinical Correlation: The Pattern of Lymph
Vessel Distribution and Diseases of the
Large Intestine | 602

Plate 71

Trachea | 692

Plate 72

Bronchioles and End Respiratory Passages | 694

Plate 73

Terminal Bronchiole, Respiratory Bronchiole,
and Alveolus | 696

Folder 17.6

Atlas Plates
Plate 54

Folder 20.2

Clinical Correlation: Antiglomerular Basement
Membrane Antibody-Induced Glomerulonephritis;
Goodpasture Syndrome | 712

Folder 20.3

Clinical Correlation: Examination of the
Urine—Urinalysis | 714

Folder 20.4

Clinical Correlation:
Renin–Angiotensin–Aldosterone System and
Hypertension | 714

72006_FM 15/07/10 7:00 PM Page xvii

Folder 20.5

Functional Considerations: Structure and
Function of Aquaporin Water Channels | 717

Folder 22.4

Clinical Correlation: Benign Prostatic
Hypertrophy and Cancer of the Prostate | 811

Folder 20.6

Functional Considerations: Hormonal
Regulation of Collecting Duct Function | 721

Folder 22.5

Clinical Correlation: Mechanism of Erection
and Erectile Dysfunction | 815

Atlas Plates
Plate 74

KIDNEY I | 728

Atlas Plates
Plate 86

Testis I | 818

Plate 75

KIDNEY II | 730

Plate 87

Testis II | 820

Plate 76

Placenta | 854
Vagina | 860
External Genitalia | 861
Mammary Glands | 863
Folder 23.1

Clinical Correlation: Polycystic Ovarian
Disease | 839

Folder 23.2

Clinical Correlation: In Vitro Fertilization | 844

Folder 23.3

Functional Considerations: Summary of
Hormonal Regulation of the Ovarian Cycle | 846

Folder 21.4

Clinical Correlation: Abnormal Thyroid Function | 758

Folder 21.5

Clinical Correlation: Chromaffin Cells and
Pheochromocytoma | 766

Folder 23.4

Clinical Correlation: Fate of the Mature

Placenta at Birth | 860

Folder 21.6

Functional Considerations: Biosynthesis
of Adrenal Hormones | 769

Folder 23.5

Clinical Correlation: Cytologic Pap Smears | 862

Folder 23.6

Clinical Correlation: Cervix and HPV
Infections | 868

Folder 23.7

Functional Considerations: Lactation and
Infertility | 870

Atlas Plates
Plate 80

Pituitary I | 772

Plate 81

Pituitary II | 774

MICROSCOPY / 13
Light Microscopy / 13
Examination of a Histologic Slide Preparation
in the Light Microscope / 14
Other Optical Systems / 15
Electron Microscopy / 18
Atomic Force Microscopy / 20
Folder 1.1 Clinical Correlation:
Frozen Sections / 4
Folder 1.2 Functional Considerations:
Feulgen Microspectrophotometry / 7
Folder 1.3 Clinical Correlation:
Monoclonal Antibodies in Medicine / 9
Folder 1.4 Proper Use of the Light
Microscope / 11

Hematoxylin and Eosin Staining With Formalin
Fixation / 2
Other Fixatives / 2
Other Staining Procedures / 3

HISTOCHEMISTRY AND
CYTOCHEMISTRY / 3
Chemical Composition of Histologic Samples / 3
Chemical Basis of Staining / 5
Enzyme Digestion / 7
Enzyme Histochemistry / 7
Immunocytochemistry / 7
Hybridization Techniques / 10
Autoradiography / 12

᭿ OVERVIEW OF METHODS USED
IN HISTOLOGY
The objective of a histology course is to lead the student
to understand the microanatomy of cells, tissues, and
organs and to correlate structure with function.
The methods used by histologists are extremely diverse.
Much of the histology course content can be framed in terms
of light microscopy. Today, students in histology laboratories
use either light microscopes or, with increasing frequency,
virtual microscopy, which represents a method of viewing a
digitized microscopic specimen on a computer screen. In the
past, more detailed interpretation of microanatomy was with
the electron microscope (EM)—both the transmission
electron microscope (TEM) and the scanning electron
microscope (SEM). Now the atomic force microscope
(AFM) can also provide high-resolution images, which are
comparable in resolution to those obtained from TEM. Both
EM and AFM, because of their greater resolution and useful
magnification, are often the last step in data acquisition from

many auxiliary techniques of cell and molecular biology.
These auxiliary techniques include:
histochemistry and cytochemistry,
immunocytochemistry and hybridization techniques,
autoradiography,
organ and tissue culture,
cell and organelle separation by differential centrifugation,
and
specialized microscopic techniques and microscopes.

•
•
•
•
•
•

The student may feel removed from such techniques and
experimental procedures because direct experience with them
is usually not available in current curricula. Nevertheless, it is
important to know something about specialized procedures
and the data they yield. This chapter provides a survey of methods and offers an explanation of how the data provided by these
methods can help the student acquire a better understanding of
cells, tissues, and organ function.
One problem students in histology face is understanding
the nature of the two-dimensional image of a histologic slide
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or an electron micrograph and how the image relates to the
three-dimensional structure from which it came. To bridge
this conceptual gap, we must first present a brief description
of the methods by which slides and electron microscopic
specimens are produced.

᭿ TISSUE PREPARATION
Hematoxylin and Eosin Staining

With Formalin Fixation
The routinely prepared hematoxylin and eosin–stained
section is the specimen most commonly studied.
The slide set given each student to study with the light microscope consists mostly of formalin-fixed, paraffin-embedded,
hematoxylin and eosin (H&E)–stained specimens. Nearly all
of the light micrographs in the Atlas section of this book are of
slides from actual student sets. Also, most photomicrographs
used to illustrate tissues and organs in histology lectures and
conferences are taken from such slides. Other staining techniques are sometimes used to demonstrate specific cell and tissue components; several of these methods are discussed below.
The first step in preparation of a tissue or organ sample is
fixation to preserve structure.
Fixation, usually by a chemical or mixture of chemicals, permanently preserves the tissue structure for subsequent treatments. Specimens should be immersed in fixative immediately
after they are removed from the body. Fixation is used to:
terminate cell metabolism,
prevent enzymatic degradation of cells and tissues by
autolysis (self-digestion),
kill pathogenic microorganisms such as bacteria, fungi,
and viruses, and
harden the tissue as a result of either cross-linking or denaturing protein molecules.

•
•
•
•

Formalin, a 37% aqueous solution of formaldehyde, at various
dilutions and in combination with other chemicals and buffers,
is the most commonly used fixative. Formaldehyde preserves
the general structure of the cell and extracellular components
by reacting with the amino groups of proteins (most often

cross-linked lysine residues). Because formaldehyde does not
significantly alter their three-dimensional structure, proteins
maintain their ability to react with specific antibodies. This
property is important in immunocytochemical staining methods (see page 7). The standard commercial solution of
formaldehyde buffered with phosphates (pH 7) acts relatively
slowly but penetrates the tissue well. However, because it does
not react with lipids, it is a poor fixative of cell membranes.

In the second step, the specimen is prepared for embedding in paraffin to permit sectioning.
Preparing a specimen for examination requires its infiltration
with an embedding medium that allows it to be thinly
sliced, typically in the range of 5 to 15 ␮m (1 micrometer
[␮m] equals 1/1,000 of a millimeter [mm]; see Table 1.1).
The specimen is washed after fixation and dehydrated in a
2

TABLE

1.1

Commonly Used Linear
Equivalents

1 picometer (pm)

ϭ

0.01 angstrom (Å)

1 angstrom

ϭ

0.1 nanometer (nm)

10 angstroms

ϭ

1.0 nanometer

1 nanometer

ϭ

1,000 picometers

1,000 nanometers

ϭ

1.0 micrometer (␮m)

1,000 micrometers

ϭ

1.0 millimeter (mm)

series of alcohol solutions of ascending concentration as high

as 100% alcohol to remove water. In the next step, clearing,
organic solvents such as xylol or toluol, which are miscible in
both alcohol and paraffin, are used to remove the alcohol before infiltration of the specimen with melted paraffin.
When the melted paraffin is cool and hardened, it is
trimmed into an appropriately sized block. The block is
then mounted in a specially designed slicing machine—a
microtome—and cut with a steel knife. The resulting sections are then mounted on glass slides using mounting
medium (pinene or acrylic resins) as an adhesive.
In the third step, the specimen is stained to permit examination.
Because paraffin sections are colorless, the specimen is not yet
suitable for light microscopic examination. To color or stain the
tissue sections, the paraffin must be dissolved out, again with
xylol or toluol, and the slide must then be rehydrated through a
series of solutions of descending alcohol concentration. The tissue on the slides is then stained with hematoxylin in water. Because the counterstain, eosin, is more soluble in alcohol than in
water, the specimen is again dehydrated through a series of alcohol solutions of ascending concentration and stained with eosin
in alcohol. Figure 1.1 shows the results of staining with hematoxylin alone, eosin alone, and hematoxylin with counterstain
eosin. After staining, the specimen is then passed through xylol
or toluol to a nonaqueous mounting medium and covered with
a coverslip to obtain a permanent preparation.

Other Fixatives
Formalin does not preserve all cell and tissue components.
Although H&E–stained sections of formalin-fixed specimens
are convenient to use because they adequately display general
structural features, they cannot elucidate the specific chemical
composition of cell components. Also, many components are
lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used.
These methods are generally based on a clear understanding of
the chemistry involved. For instance, the use of alcohols and
organic solvents in routine preparations removes neutral lipids.

To retain neutral lipids, such as those in adipose cells, frozen
sections of formalin-fixed tissue and dyes that dissolve in fats
must be used; to retain membrane structures, special fixatives

72006_ch01 15/07/10 2:08 PM Page 3

chapter 1

a

b

c

3

containing heavy metals that bind to the phospholipids, such
as permanganate and osmium, are used (Folder 1.1). The routine use of osmium tetroxide as a fixative for electron microscopy is the primary reason for the excellent preservation of
membranes in electron micrographs.

Other Staining Procedures
Hematoxylin and eosin are used in histology primarily to
display structural features.
Despite the merits of H&E staining, the procedure does not
adequately reveal certain structural components of histologic
sections such as elastic material, reticular fibers, basement
membranes, and lipids. When it is desirable to display these
components, other staining procedures, most of them selective, can be used. These procedures include the use of orcein
and resorcin-fuchsin for elastic material and silver impregnation for reticular fibers and basement membrane material. Although the chemical bases of many staining methods are not

always understood, they work. Knowing the components that
a procedure reveals is more important than knowing precisely
how the procedure works.

᭿ HISTOCHEMISTRY AND
CYTOCHEMISTRY
Specific chemical procedures can provide information
about the function of cells and the extracellular components of tissues.
Histochemical and cytochemical procedures may be based on
specific binding of a dye, use of a fluorescent dye–labeled

antibody with a particular cell component, or the inherent
enzymatic activity of a cell component. In addition, many

large molecules found in cells can be localized by the process
of autoradiography, in which radioactively tagged precursors of the molecule are incorporated by cells and tissues before fixation. Many of these procedures can be used with both
light microscopic and electron microscopic preparations.
Before discussing the chemistry of routine staining and
histochemical and cytochemical methods, it is useful to examine briefly the nature of a routinely fixed and embedded
section of a specimen.

Chemical Composition of Histologic
Samples
The chemical composition of a tissue ready for routine
staining differs from living tissue.
The components that remain after fixation consist mostly of
large molecules that do not readily dissolve, especially after
treatment with the fixative. These large molecules, particularly those that react with other large molecules to form
macromolecular complexes, are usually preserved in a tissue
section. Examples of such large macromolecular complexes

include:
nucleoproteins formed from nucleic acids bound to
protein,
intracellular cytoskeletal proteins complexed with associated proteins,
extracellular proteins in large insoluble aggregates,
bound to similar molecules by cross-linking of neighboring molecules, as in collagen fiber formation, and

•
•
•

3

Methods ᭹ H I STO C H E M I STRY AN D CYTO C H E M I STRY

FIGURE 1.1 • Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections that
demonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. a. This photomicrograph
reveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structures
that have a high affinity for the dye are most heavily stained−for example, the nuclear DNA and areas of the cell containing cytoplasmic
RNA. b. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, that
the nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin and
then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those
components to which it has a high affinity. c. This photomicrograph reveals the combined staining effect of H&E. ϫ480.

72006_ch01 15/07/10 2:08 PM Page 4

• FOLDER 1.1

Clinical Correlation: Frozen Sections

Sometimes, the pathologist may be asked to immediately
evaluate tissue obtained during surgery, especially when instant pathologic diagnosis may determine how the surgery
will proceed. There are several indications to perform such
an evaluation, routinely known as a frozen section. Most
commonly, a surgeon in the operating room requests a
frozen section when no preoperative diagnosis was available
or when unexpected intraoperative findings must be identified. In addition, the surgeon may want to know whether all of
a pathologic mass within the healthy tissue limit has been removed and whether the margin of the surgical resection is
free of diseased tissue. Frozen sections are also done in
combination with other procedures such as endoscopy or
thin-needle biopsy to confirm whether the obtained biopsy
material will be usable in further pathologic examinations.
Three main steps are involved in frozen section preparation:
the tissue sample. Small tissue samples are
• Freezing
frozen either by using compressed carbon dioxide or by
immersion in a cold fluid (isopentane) at a temperature of

Ϫ50ЊC. Freezing can be achieved in a special highefficiency refrigerator. Freezing makes the tissue solid
and allows sectioning with a microtome.
the frozen tissue. Sectioning is usually per• Sectioning
formed inside a cryostat, a refrigerated compartment
containing a microtome. Because the tissue is frozen
solid, it can be cut into extremely thin (5 to 10 ␮m) sections. The sections are then mounted on glass slides.
the cut sections. Staining is done to differen• Staining
tiate cell nuclei from the rest of the tissue. The most
common stains used for frozen sections are H&E,
methylene blue (Fig. F1.1.1), and PAS stains.
The entire process of preparation and evaluation of frozen

sections may take as little as 10 minutes to complete. The
total time to obtain results largely depends on the transport
time of the tissue from the operating room to the pathology
laboratory, on the pathologic technique used, and the experience of the pathologist. The findings are then directly communicated to the surgeon waiting in the operating room.

F1.1.1 • Evaluation of a
specimen obtained during surgery
by frozen-section technique. a. This
photomicrograph shows a specimen
obtained from the large intestine that
was prepared by frozen-section
technique and stained with methylene
blue. ϫ160 b. Part of the specimen
was fixed in formalin and processed as
a routine H&E preparation. Examination
of the frozen section revealed it to be
normal. This diagnosis was later
confirmed by examining the routinely
prepared H&E specimen. ϫ180.
(Courtesy of Dr. Daniel W. Visscher.)

FIGURE

a
phospholipid–protein (or carbohydrate)
• membrane
complexes.

These molecules constitute the structure of cells and tissues—
that is, they make up the formed elements of the tissue. They

are the basis for the organization that is seen in tissue with the
microscope.
In many cases, a structural element is also a functional
unit. For example, in the case of proteins that make up the
contractile filaments of muscle cells, the filaments are the visible structural components and the actual participants in the
contractile process. The RNA of the cytoplasm is visualized as
4

b

part of a structural component (e.g., ergastoplasm of secretory cells, Nissl bodies of nerve cells) and is also the actual
participant in the synthesis of protein.
Many tissue components are lost during the routine
preparation of H&E–stained sections.
Despite the fact that nucleic acids, proteins, and phospholipids are mostly retained in tissue sections, many are also
lost. Small proteins and small nucleic acids, such as transfer
RNA, are generally lost during the preparation of the tissue.
As previously described, neutral lipids are usually dissolved by
the organic solvents used in tissue preparation. Other large

72006_ch01 15/07/10 2:08 PM Page 5

molecules also may be lost, for example, by being hydrolyzed
because of the unfavorable pH of the fixative solutions. Examples of large molecules lost during routine fixation in
aqueous fixatives are:
glycogen (an intracellular storage carbohydrate common
in liver and muscle cells), and
proteoglycans and glycosaminoglycans (extracellular
complex carbohydrates found in connective tissue).

TABLE

1.2

Dye

•
•

Some Basic and Acidic
Dyes
Color

Basic dyes

Methylene blue

Blue

These molecules can be preserved, however, by using a nonaqueous fixative for glycogen or by adding specific binding
agents to the fixative solution that preserve extracellular
carbohydrate-containing molecules.

Pyronin G

Red

Toluidine blue

Blue

Soluble components, ions, and small molecules are also
lost during the preparation of paraffin sections.

Acid fuchsin

Red

Aniline blue

Blue

Eosin

Red

Orange G

Orange

Intermediary metabolites, glucose, sodium, chloride, and
similar substances are lost during preparation of routine
H&E paraffin sections. Many of these substances can
be studied in special preparations, sometimes with considerable loss of structural integrity. These small soluble ions and
molecules do not make up the formed elements of a tissue;
they participate in synthetic processes or cellular reactions.
When they can be preserved and demonstrated by specific
methods, they provide invaluable information about cell
metabolism, active transport, and other vital cellular processes. Water, a highly versatile molecule, participates in these

reactions and processes and contributes to the stabilization of
macromolecular structure through hydrogen bonding.

Acidic dyes

5

acidic to neutral pH (5 to 7), sulfate and phos• Atphatea slightly
groups are ionized and available for reaction with the

•

basic dye by electrostatic linkages.
At low pH (below 4), only sulfate groups remain ionized
and react with basic dyes.

An acidic dye, such as eosin, carries a net negative charge on
its colored portion and is described by the general formula
[NaϩdyeϪ].
A basic dye carries a net positive charge on its colored portion and is described by the general formula [dyeϩClϪ].
Hematoxylin does not meet the definition of a strict basic
dye but has properties that closely resemble those of a basic
dye. The color of a dye is not related to whether it is basic or
acidic, as can be noted by the examples of basic and acidic
dyes listed in Table 1.2.

Therefore, staining with basic dyes at a specific pH can be used
to focus on specific anionic groups; because the specific anionic
groups are found predominantly on certain macromolecules,
the staining serves as an indicator of these macromolecules.

As mentioned, hematoxylin is not, strictly speaking, a
basic dye. It is used with a mordant (i.e., an intermediate
link between the tissue component and the dye). The mordant causes the stain to resemble a basic dye. The linkage in
the tissue–mordant–hematoxylin complex is not a simple electrostatic linkage; when sections are placed in water,
hematoxylin does not dissociate from the tissue. Hematoxylin lends itself to those staining sequences in which it is
followed by aqueous solutions of acidic dyes. True basic
dyes, as distinguished from hematoxylin, are not generally
used in sequences in which the basic dye is followed by an
acidic dye. The basic dye then tends to dissociate from the
tissue during the aqueous solution washes between the two
dye solutions.

Basic dyes react with anionic components of cells and
tissue (components that carry a net negative charge).

Acidic dyes react with cationic groups in cells and tissues,
particularly with the ionized amino groups of proteins.

Anionic components include the phosphate groups of nucleic acids, the sulfate groups of glycosaminoglycans, and the
carboxyl groups of proteins. The ability of such anionic groups
to react with a basic dye is called basophilia [Gr., base-loving].
Tissue components that stain with hematoxylin also exhibit
basophilia.
The reaction of the anionic groups varies with pH. Thus:
At a high pH (about 10), all three groups are ionized and available for reaction by electrostatic linkages with the basic dye.

The reaction of cationic groups with an acidic dye is called
acidophilia [Gr., acid-loving]. Reactions of cell and tissue
components with acidic dyes are neither as specific nor as precise as reactions with basic dyes.
Although electrostatic linkage is the major factor in the primary binding of an acidic dye to the tissue, it is not the only

one; because of this, acidic dyes are sometimes used in combinations to color different tissue constituents selectively. For example, three acidic dyes are used in the Mallory staining

Chemical Basis of Staining
Acidic and Basic Dyes
Hematoxylin and eosin are the most commonly used dyes
in histology.

•

5

Methods ᭹ H I STO C H E M I STRY AN D CYTO C H E M I STRY

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