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 CONTENTS i

FIFTEENTH EDITION

Junqueira’s

Basic Histology T E X T A N D AT L A S

Anthony L. Mescher, PhD

Professor of Anatomy and Cell Biology
Indiana University School of Medicine
Bloomington, Indiana

New York Chicago San Francisco Athens London Madrid Mexico City
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00_Mescher_FM_pi-x.indd 1 27/04/18 11:03 am

Copyright © 2018 by McGraw-Hill Education. All rights reserved. Except as permitted under the United States Copyright Act of 1976,
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 Contents
PREFACE VII | ACKNOWLEDGMENTS IX

1 Histology & Its Methods 5 Connective Tissue 96

of Study 1 Cells of Connective Tissue 96
Preparation of Tissues for Study 1 Fibers 103
Light Microscopy 4 Ground Substance 111
Electron Microscopy 8 Types of Connective Tissue 114
Autoradiography 9 Summary of Key Points 119
Cell & Tissue Culture 10 Assess Your Knowledge 120
Enzyme Histochemistry 10
Visualizing Specific Molecules 10 6 Adipose Tissue 122
Interpretation of Structures in Tissue White Adipose Tissue 122
Sections 14 Brown Adipose Tissue 126
Summary of Key Points 15 Summary of Key Points 127
Assess Your Knowledge 16 Assess Your Knowledge 128

2 The Cytoplasm 17 7 Cartilage 129

Cell Differentiation 17 Hyaline Cartilage 129
The Plasma Membrane 17 Elastic Cartilage 133
Cytoplasmic Organelles 27 Fibrocartilage 134
The Cytoskeleton 42 Cartilage Formation, Growth, & Repair 134
Inclusions 47 Summary of Key Points 136
Summary of Key Points 51 Assess Your Knowledge 136
Assess Your Knowledge 52
8 Bone 138
3 The Nucleus 53 Bone Cells 138
Components of the Nucleus 53 Bone Matrix 143
The Cell Cycle 58 Periosteum & Endosteum 143
Mitosis 61 Types of Bone 143
Stem Cells & Tissue Renewal 65 Osteogenesis 148
Meiosis 65 Bone Remodeling & Repair 152
Apoptosis 67 Metabolic Role of Bone 153
Summary of Key Points 69 Joints 155
Assess Your Knowledge 70 Summary of Key Points 158
Assess Your Knowledge 159
4 Epithelial Tissue 71
Characteristic Features of Epithelial Cells 72 9 Nerve Tissue & the Nervous
Specializations of the Apical Cell Surface 77 System 161
Types of Epithelia 80 Development of Nerve Tissue 161
Transport Across Epithelia 88 Neurons 163
Renewal of Epithelial Cells 88 Glial Cells & Neuronal Activity 168
Summary of Key Points 90 Central Nervous System 175
Assess Your Knowledge 93 Peripheral Nervous System 182

iii

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 iv CONTENTS

Neural Plasticity & Regeneration 187 15 Digestive Tract 295

Summary of Key Points 190
General Structure of the Digestive Tract 295
Assess Your Knowledge 191
Oral Cavity 298
Esophagus 305
10 Muscle Tissue 193 Stomach 307
Skeletal Muscle 193 Small Intestine 314
Cardiac Muscle 207 Large Intestine 318
Smooth Muscle 208 Summary of Key Points 326
Regeneration of Muscle Tissue 213 Assess Your Knowledge 327
Summary of Key Points 213
Assess Your Knowledge 214 16 Organs Associated with the Digestive
Tract 329
11 The Circulatory System 215 Salivary Glands 329
Heart 215 Pancreas 332
Tissues of the Vascular Wall 219 Liver 335
Vasculature 220 Biliary Tract & Gallbladder 345
Lymphatic Vascular System 231 Summary of Key Points 346
Summary of Key Points 235 Assess Your Knowledge 348
Assess Your Knowledge 235

17 The Respiratory System 349

12 Blood 237 Nasal Cavities 349
Composition of Plasma 237 Pharynx 352
Blood Cells 239 Larynx 352
Summary of Key Points 250 Trachea 354
Assess Your Knowledge 252 Bronchial Tree & Lung 354
Lung Vasculature & Nerves 366
13 Hemopoiesis 254 Pleural Membranes 368
Stem Cells, Growth Factors, & Differentiation 254 Respiratory Movements 368
Bone Marrow 255 Summary of Key Points 369
Maturation of Erythrocytes 258 Assess Your Knowledge 369
Maturation of Granulocytes 260
Maturation of Agranulocytes 263 18 Skin 371
Origin of Platelets 263 Epidermis 372
Summary of Key Points 265 Dermis 378
Assess Your Knowledge 265 Subcutaneous Tissue 381
Sensory Receptors 381
14 The Immune System & Lymphoid Hair 383
Organs 267 Nails 384
Innate & Adaptive Immunity 267 Skin Glands 385
Cytokines 269 Skin Repair 388
Antigens & Antibodies 270 Summary of Key Points 391
Antigen Presentation 271 Assess Your Knowledge 391
Cells of Adaptive Immunity 273
Thymus 276 19 The Urinary System 393
Mucosa-Associated Lymphoid Tissue 281 Kidneys 393
Lymph Nodes 282 Blood Circulation 394
Spleen 286 Renal Function: Filtration, Secretion, &
Summary of Key Points 293 Reabsorption 395
Assess Your Knowledge 294 Ureters, Bladder, & Urethra 406

00_Mescher_FM_pi-x.indd 4 27/04/18 11:03 am


Summary of Key Points 411
Assess Your Knowledge 412
20 Endocrine Glands 413
Pituitary Gland (Hypophysis) 413
Adrenal Glands 423
Pancreatic Islets 427
Diffuse Neuroendocrine System 429
Thyroid Gland 429
Parathyroid Glands 432
Pineal Gland 434
Summary of Key Points 437
Assess Your Knowledge 437
21 The Male Reproductive
System 439
Testes 439
Intratesticular Ducts 449
Excretory Genital Ducts 450
Accessory Glands 451
Penis 456
Summary of Key Points 457
Assess Your Knowledge 459
00_Mescher_FM_pi-x.indd 5
CONTENTS v
22 The Female Reproductive System 460
Ovaries 460
Uterine Tubes 470
Major Events of Fertilization 471
Uterus 471
Embryonic Implantation, Decidua, & the Placenta 478
Cervix 482
Vagina 483
External Genitalia 483
Mammary Glands 483
Summary of Key Points 488
Assess Your Knowledge 489
23 The Eye & Ear: Special Sense
Organs 490
Eyes: The Photoreceptor System 490
Ears: The Vestibuloauditory System 509
Summary of Key Points 522
Assess Your Knowledge 522
APPENDIX 525
FIGURE CREDITS 527
INDEX 529
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 Preface
With this 15th edition, Junqueira’s Basic Histology continues as
the preeminent source of concise yet thorough information
on human tissue structure and function. For over 45 years
this educational resource has met the needs of learners for a
well-organized and concise presentation of cell biology and
histology that integrates the material with that of biochemistry,
immunology, endocrinology, and physiology and provides
an excellent foundation for subsequent studies in pathology.
The text is prepared specifically for students of medicine
and other health-related professions, as well as for advanced
undergraduate courses in tissue biology. As a result of its value
and appeal to students and instructors alike, Junqueira’s Basic
Histology has been translated into a dozen different languages
and is used by medical students throughout the world.
Unlike other histology texts and atlases, the present
work again includes with each chapter a set of multiple-
choice Self-Test Questions that allow readers to assess their
comprehension and knowledge of important points in that
chapter. At least a few questions in each set utilize clinical
vignettes or cases to provide context for framing the medical
relevance of concepts in basic science, as recommended by the US
National Board of Medical Examiners. As with the last edition,
each chapter also includes a Summary of Key Points designed to
guide the students concerning what is clearly important and what
is less so. Summary Tables in each chapter organize and condense
important information, further facilitating efficient learning.
Each chapter has been revised and shortened, while
coverage of specific topics has been expanded and updated as
needed. Study is facilitated by modern page design. Inserted
throughout each chapter are more numerous, short paragraphs
that indicate how the information presented can be used
medically and which emphasize the foundational relevance of
the material learned.
The art and other figures are presented in each chapter,
with the goal to simplify learning and integration with
related material. The McGraw-Hill medical illustrations,
now used throughout the text, are the most useful, thorough,
and attractive of any similar medical textbook. Electron and
light micrographs have been replaced throughout the book
as needed, and they again make up a complete atlas of cell,
tissue, and organ structures fully compatible with the students’
own collection of glass or digital slides. Health science
students whose medical library offers AccessMedicine among
its electronic resources (which includes more than 95% of
00_Mescher_FM_pi-x.indd 7
U.S. medical schools) can access a complete human histology
Laboratory Guide linked to the virtual microscope at the URL
given below. This digital Laboratory Guide, which is new with
this edition of the text and unique among learning resources
offered by any histology text and atlas, provides both links
to the appropriate microscope slides needed for each topic
and links to the correlated figures or tables in the text. Those
without AccessMedicine will lack the digital Laboratory Guide,
but may still study and utilize the 150 virtual microscope
slides of all human tissues and organs, which are available at:
http://medsci.indiana.edu/junqueira/virtual/junqueira.htm.
As with the previous edition, the book facilitates learning
by its organization:
■ An opening chapter reviews the histological techniques
that allow understanding of cell and tissue structure.
■ Two chapters then summarize the structural and
functional organization of human cell biology,
presenting the cytoplasm and nucleus separately.
■ The next seven chapters cover the four basic tissues that
make up our organs: epithelia, connective tissue (and its
major sub-types), nervous tissue, and muscle.
■ Remaining chapters explain the organization and
functional significance of these tissues in each of
the body’s organ systems, closing with up-to-date
consideration of cells in the eye and ear.
For additional review of what’s been learned or to
assist rapid assimilation of the material in Junqueira’s Basic
Histology, McGraw-Hill has published a set of 200 full-color
Basic Histology Flash Cards, also authored by Anthony
Mescher. Each card includes images of key structures to
identify, a summary of important facts about those structures,
and a clinical comment. This valuable learning aid is available
as a set of actual cards from Amazon.com, or as an app for
smartphones or tablets from the online App Store.
With its proven strengths and the addition of new features,
I am confident that Junqueira’s Basic Histology will continue
as one of the most valuable and most widely read educational
resources in histology. Users are invited to provide feedback
to the author with regard to any aspect of the book’s features.
Anthony L. Mescher
Indiana University School of Medicine
mescher@indiana.edu
vii
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 Acknowledgments
I wish to thank the students at Indiana University School of
Medicine and the undergraduates at Indiana University with
whom I have studied histology and cell biology for over 35 years
and from whom I have learned much about presenting basic
concepts most effectively. Their input has greatly helped in the
task of maintaining and updating the presentations in this classic
textbook. As with the last edition, the help of Sue Childress and
Dr. Mark Braun was invaluable in slide preparation and the
virtual microscope for human histology respectively.
As with the last edition, the present text includes ten
multiple-choice questions at the end of each chapter, aimed
to test the learner’s retention and understanding of important
points in that body of material. Many of these questions
were used in my courses, but others are taken or modified
from a few of the many excellent review books published by
McGraw-Hill/Lange for students preparing to take the U.S.
Medical Licensing Examination. These include Histology and
Cell Biology: Examination and Board Review, by Douglas
Paulsen; USMLE Road Map: Histology, by Harold Sheedlo; and
Anatomy, Histology, & Cell Biology: PreTest Self-Assessment &
00_Mescher_FM_pi-x.indd 9
Review, by Robert Klein and George Enders. The use here
of questions from these valuable resources is gratefully
acknowledged. Students are referred to those review books for
hundreds of additional self-assessment questions.
I am also grateful to my colleagues and reviewers from
throughout the world who provided specialized expertise or
original photographs, which are also acknowledged in figure
captions. I thank those professors and students in the United
States and countries throughout the world who provided useful
suggestions that have improved the new edition of Junqueira’s
Basic Histology. Finally, I am pleased to acknowledge the help
and collegiality provided by the staff of McGraw-Hill, especially
editors Michael Weitz and Brian Kearns, whose work made
possible publication of this 15th edition of Junqueira’s Basic
Histology.
Anthony L. Mescher
Indiana University School of Medicine
mescher@indiana.edu
ix
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 C H A P T E R

Fixation
1
PREPARATION OF TISSUES FOR STUDY
Histology & Its
Methods of Study
1
1
AUTORADIOGRAPHY 9
CELL & TISSUE CULTURE 10
Embedding & Sectioning 3
ENZYME HISTOCHEMISTRY 10
Staining 3
LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10
Bright-Field Microscopy 4 Immunohistochemistry 11
Fluorescence Microscopy 5 Hybridization Techniques 12
Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE
Confocal Microscopy 5 SECTIONS 14
Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15
ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16
Transmission Electron Microscopy 8
Scanning Electron Microscopy 9

H istology is the study of the tissues of the body and

how these tissues are arranged to constitute organs.
This subject involves all aspects of tissue biology, with
the focus on how cells’ structure and arrangement optimize
functions specific to each organ.
Tissues have two interacting components: cells and extra-
cellular matrix (ECM). The ECM consists of many kinds of
macromolecules, most of which form complex structures,
such as collagen fibrils. The ECM supports the cells and con-
tains the fluid transporting nutrients to the cells, and carry-
ing away their wastes and secretory products. Cells produce
the ECM locally and are in turn strongly influenced by matrix
molecules. Many matrix components bind to specific cell
surface receptors that span the cell membranes and connect
to structural components inside the cells, forming a contin-
uum in which cells and the ECM function together in a well-
coordinated manner.
During development, cells and their associated matrix
become functionally specialized and give rise to fundamen-
tal types of tissues with characteristic structural features.
Organs are formed by an orderly combination of these tissues,
and their precise arrangement allows the functioning of each
organ and of the organism as a whole.
The small size of cells and matrix components makes his-
tology dependent on the use of microscopes and molecular
methods of study. Advances in biochemistry, molecular biol-
ogy, physiology, immunology, and pathology are essential for
a better knowledge of tissue biology. Familiarity with the tools
and methods of any branch of science is essential for a proper
understanding of the subject. This chapter reviews common
methods used to study cells and tissues, focusing on micro-
scopic approaches.
››PREPARATION OF TISSUES
FOR STUDY
The most common procedure used in histologic research is
the preparation of tissue slices or “sections” that can be exam-
ined visually with transmitted light. Because most tissues and
organs are too thick for light to pass through, thin translu-
cent sections are cut from them and placed on glass slides for
microscopic examination of the internal structures.
The ideal microscopic preparation is preserved so that the
tissue on the slide has the same structural features it had in the
body. However, this is often not feasible because the prepara-
tion process can remove cellular lipid, with slight distortions
of cell structure. The basic steps used in tissue preparation for
light microscopy are shown in Figure 1–1.
Fixation
To preserve tissue structure and prevent degradation by
enzymes released from the cells or microorganisms, pieces of

01_Mescher_ch01_p001-016.indd 1 27/04/18 6:44 pm

 2 CHAPTER 1 ■ Histology & Its Methods of Study
FIGURE 1–1 Sectioning fixed and embedded tissue.
(a) Fixation Dehydration
Most tissues studied histologically are prepared as shown, with
this sequence of steps (a):
■■ Fixation: Small pieces of tissue are placed in solutions of
chemicals that cross-link proteins and inactivate degradative
enzymes, which preserve cell and tissue structure.
■■ Dehydration: The tissue is transferred through a series of
increasingly concentrated alcohol solutions, ending in 100%,
which removes all water.
■■ Clearing: Alcohol is removed in organic solvents in which
both alcohol and paraffin are miscible.
■■ Infiltration: The tissue is then placed in melted paraffin until it
becomes completely infiltrated with this substance.
■■ Embedding: The paraffin-infiltrated tissue is placed in a small
mold with melted paraffin and allowed to harden.
■■ Trimming: The resulting paraffin block is trimmed to expose
the tissue for sectioning (slicing) on a microtome.
organs are placed as soon as possible after removal from the
body in solutions of stabilizing or cross-linking compounds
called fixatives. Because a fixative must fully diffuse through
the tissues to preserve all cells, tissues are usually cut into
small fragments before fixation to facilitate penetration. To
improve cell preservation in large organs, fixatives are often
introduced via blood vessels, with vascular perfusion allowing
fixation rapidly throughout the tissues.
One widely used fixative for light microscopy is forma-
lin, a buffered isotonic solution of 37% formaldehyde. Both
this compound and glutaraldehyde, a fixative used for electron
01_Mescher_ch01_p001-016.indd 2
52°- 60°C
Clearing Infiltration Embedding
Drive wheel
Block holder
Paraffin block
Tissue
Steel knife
Similar steps are used in preparing tissue for transmission elec-
tron microscopy (TEM), except special fixatives and dehydrating
solutions are used with smaller tissue samples and embedding
involves epoxy resins which become harder than paraffin to allow
very thin sectioning.
(b) A microtome is used for sectioning paraffin-embedded tissues
for light microscopy. The trimmed tissue specimen is mounted
in the paraffin block holder, and each turn of the drive wheel by
the histologist advances the holder a controlled distance, gener-
ally from 1 to 10 μm. After each forward move, the tissue block
passes over the steel knife edge and a section is cut at a thickness
equal to the distance the block advanced. The paraffin sections
are placed on glass slides and allowed to adhere, deparaffinized,
and stained for light microscope study. For TEM, sections less than
1 μm thick are prepared from resin-embedded cells using an ultra-
microtome with a glass or diamond knife.
microscopy, react with the amine groups (NH2) of proteins,
preventing their degradation by common proteases. Glutaral-
dehyde also cross-links adjacent proteins, reinforcing cell and
ECM structures.
Electron microscopy provides much greater magni-
fication and resolution of very small cellular structures,
and fixation must be done very carefully to preserve addi-
tional “ultrastructural” detail. Typically in such studies,
glutaraldehyde-treated tissue is then immersed in buffered
osmium tetroxide, which preserves (and stains) cellular lip-
ids as well as proteins.
26/04/18 11:10 am

Embedding & Sectioning
To permit thin sectioning, fixed tissues are infiltrated and
embedded in a material that imparts a firm consistency.
Embedding materials include paraffin, used routinely for light
microscopy, and plastic resins, which are adapted for both
light and electron microscopy.
Before infiltration with such media, the fixed tissue must
undergo dehydration by having its water extracted gradually
by transfers through a series of increasing ethanol solutions,
ending in 100% ethanol. The ethanol is then replaced by an
organic solvent miscible with both alcohol and the embedding
medium, a step referred to as clearing because infiltration with
the reagents used here gives the tissue a translucent appearance.
The fully cleared tissue is then placed in melted paraffin
in an oven at 52°-60°C, which evaporates the clearing solvent
and promotes infiltration of the tissue with paraffin, and then
embedded by allowing it to harden in a small container of
paraffin at room temperature. Tissues to be embedded with
plastic resin are also dehydrated in ethanol and then infiltrated
with plastic solvents that harden when cross-linking polymer-
izers are added. Plastic embedding avoids the higher tempera-
tures needed with paraffin, which helps avoid tissue distortion.
The hardened block with tissue and surrounding embed-
ding medium is trimmed and placed for sectioning in an
instrument called a microtome (see Figure 1–1). Paraffin sec-
tions are typically cut at 3-10 μm thickness for light microscopy,
but electron microscopy requires sections less than 1 μm thick.
One micrometer (1 μm) equals 1/1000 of a millimeter (mm)
or 10−6 m. Other spatial units commonly used in microscopy
are the nanometer (1 nm = 0.001 μm = 10−6 mm = 10−9 m) and
angstrom (1 Å = 0.1 nm or 10−4 μm). The sections are placed on
glass slides and stained for light microscopy or on metal grids
for electron-microscopic staining and examination.
› ›› MEDICAL APPLICATION
Biopsies are tissue samples removed during surgery or routine
medical procedures. In the operating room, biopsies are fixed
in vials of formalin for processing and microscopic analysis in
a pathology laboratory. If results of such analyses are required
before the medical procedure is completed, for example to
know whether a growth is malignant before the patient is
closed, a much more rapid processing method is used. The
biopsy is rapidly frozen in liquid nitrogen, preserving cell
structures and at the same time making the tissue hard and
ready for sectioning. A microtome called a cryostat in a cabi-
net at subfreezing temperature is used to section the block
with tissue, and the frozen sections are placed on slides for
rapid staining and microscopic examination by a pathologist.
Freezing of tissues is also effective in histochemical stud-
ies of very sensitive enzymes or small molecules because
freezing, unlike fixation, does not inactivate most enzymes.
Finally, because clearing solvents often dissolve cell lipids in
fixed tissues, frozen sections are also useful when structures
containing lipids are to be studied histologically.
01_Mescher_ch01_p001-016.indd 3
Preparation of Tissues for Study 3
Staining
C H A P T E R
Most cells and extracellular material are completely color-
less, and to be studied microscopically tissue sections must
be stained (dyed). Methods of staining have been devised that
make various tissue components not only conspicuous but also
distinguishable from one another. Dyes stain material more or
less selectively, often behaving like acidic or basic compounds
and forming electrostatic (salt) linkages with ionizable radicals
1
of macromolecules in tissues. Cell components, such as nucleic
Histology & Its Methods of Study ■ Preparation of Tissues for Study
acids with a net negative charge (anionic), have an affinity for
basic dyes and are termed basophilic; cationic components,
such as proteins with many ionized amino groups, stain more
readily with acidic dyes and are termed acidophilic.
Examples of basic dyes include toluidine blue, alcian blue,
and methylene blue. Hematoxylin behaves like a basic dye,
staining basophilic tissue components. The main tissue com-
ponents that ionize and react with basic dyes do so because of
acids in their composition (DNA, RNA, and glycosaminogly-
cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain
the acidophilic components of tissues such as mitochondria,
secretory granules, and collagen.
Of all staining methods, the simple combination of
hematoxylin and eosin (H&E) is used most commonly.
Hematoxylin stains DNA in the cell nucleus, RNA-rich por-
tions of the cytoplasm, and the matrix of cartilage, produc-
ing a dark blue or purple color. In contrast, eosin stains other
cytoplasmic structures and collagen pink (Figure 1–2a). Here
eosin is considered a counterstain, which is usually a single
dye applied separately to distinguish additional features of a
tissue. More complex procedures, such as trichrome stains (eg,
Masson’s trichrome), allow greater distinctions among various
extracellular tissue components.
The periodic acid–Schiff (PAS) reaction utilizes the
hexose rings of polysaccharides and other carbohydrate-rich
tissue structures and stains such macromolecules distinctly
purple or magenta. Figure 1–2b shows an example of cells with
carbohydrate-rich areas well-stained by the PAS reaction. The
DNA of cell nuclei can be specifically stained using a modifica-
tion of the PAS procedure called the Feulgen reaction.
Basophilic or PAS-positive material can be further identi-
fied by enzyme digestion, pretreatment of a tissue section with
an enzyme that specifically digests one substrate. For example,
pretreatment with ribonuclease will greatly reduce cytoplas-
mic basophilia with little overall effect on the nucleus, indicat-
ing the importance of RNA for the cytoplasmic staining.
Lipid-rich structures of cells are revealed by avoiding the
processing steps that remove lipids, such as treatment with
heat and organic solvents, and staining with lipid-soluble
dyes such as Sudan black, which can be useful in diagnosis
of metabolic diseases that involve intracellular accumulations
of cholesterol, phospholipids, or glycolipids. Less common
methods of staining can employ metal impregnation tech-
niques, typically using solutions of silver salts to visual certain
ECM fibers and specific cellular elements in nervous tissue.
The Appendix lists important staining procedures used for
most of the light micrographs in this book.
26/04/18 11:10 am
 4 CHAPTER 1 ■ Histology & Its Methods of Study
FIGURE 1–2 Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining.
G G
G
L
a b
Micrographs of epithelium lining the small intestine, (a) stained
with H&E, and (b) stained with the PAS reaction for glycoproteins.
With H&E, basophilic cell nuclei are stained purple, while cyto-
plasm stains pink. Cell regions with abundant oligosaccharides
on glycoproteins, such as the ends of the cells at the lumen (L)
or the scattered mucus-secreting goblet cells (G), are poorly
stained. With PAS, however, cell staining is most intense at the
Slide preparation, from tissue fixation to observation
with a light microscope, may take from 12 hours to 2½ days,
depending on the size of the tissue, the embedding medium,
and the method of staining. The final step before microscopic
observation is mounting a protective glass coverslip on the
slide with clear adhesive.
››LIGHT MICROSCOPY
Conventional bright-field microscopy and more specialized
applications like fluorescence, phase-contrast, confocal, and
polarizing microscopy are all based on the interaction of light
with tissue components and are used to reveal and study tissue
features.
Bright-Field Microscopy
With the bright-field microscope, stained tissue is examined
with ordinary light passing through the preparation. As shown
in Figure 1–3, the microscope includes an optical system and
mechanisms to move and focus the specimen. The optical
components are the condenser focusing light on the object
to be studied; the objective lens enlarging and projecting the
image of the object toward the observer; and the eyepiece
01_Mescher_ch01_p001-016.indd 4
L
lumen, where projecting microvilli have a prominent layer of
glycoproteins at the lumen (L) and in the mucin-rich secretory
granules of goblet cells. Cell surface glycoproteins and mucin are
PAS-positive because of their high content of oligosaccharides
and polysaccharides, respectively. The PAS-stained tissue was
counterstained with hematoxylin to show the cell nuclei.
(a. X400; b. X300)
(or ocular lens) further magnifying this image and projecting
it onto the viewer’s retina or a charge-coupled device (CCD)
highly sensitive to low light levels with a camera and monitor.
The total magnification is obtained by multiplying the magni-
fying power of the objective and ocular lenses.
The critical factor in obtaining a crisp, detailed image
with a light microscope is its resolving power, defined as the
smallest distance between two structures at which they can be
seen as separate objects. The maximal resolving power of the
light microscope is approximately 0.2 μm, which can permit
clear images magnified 1000-1500 times. Objects smaller or
thinner than 0.2 μm (such as a single ribosome or cytoplasmic
microfilament) cannot be distinguished with this instrument.
Likewise, two structures such as mitochondria will be seen as
only one object if they are separated by less than 0.2 μm. The
microscope’s resolving power determines the quality of the
image, its clarity and richness of detail, and depends mainly on
the quality of its objective lens. Magnification is of value only
when accompanied by high resolution. Objective lenses pro-
viding higher magnification are designed to also have higher
resolving power. The eyepiece lens only enlarges the image
obtained by the objective and does not improve resolution.
Virtual microscopy, typically used for study of bright-
field microscopic preparations, involves the conversion of a
26/04/18 11:10 am
 Light Microscopy 5

FIGURE 1–3 Components and light path of a

Fluorescence Microscopy

C H A P T E R
bright-field microscope. When certain cellular substances are irradiated by light of a
proper wavelength, they emit light with a longer wavelength—
Eyepiece
Interpupillar
adjustment
a phenomenon called fluorescence. In fluorescence
Binocular
tubes Head microscopy, tissue sections are usually irradiated with ultra-
violet (UV) light and the emission is in the visible portion of
the spectrum. The fluorescent substances appear bright on
Stand
a dark background. For fluorescent microscopy, the instru-

1
Measuring
ment has a source of UV or other light and filters that select

Histology & Its Methods of Study ■ Light Microscopy

graticule
Beamsplitter
rays of different wavelengths emitted by the substances to be
Revolving
nosepiece visualized.
Specimen Objective Fluorescent compounds with affinity for specific cell
holder macromolecules may be used as fluorescent stains. Acridine
Mechanical
stage orange, which binds both DNA and RNA, is an example.
On/off
switch When observed in the fluorescence microscope, these nucleic
Condenser Illumination
intensity
acids emit slightly different fluorescence, allowing them to be
Field lens
control localized separately in cells (Figure 1–4a). Other compounds,
Field such as DAPI and Hoechst, stain specifically bind DNA and
diaphragm
Collector
are used to stain cell nuclei, emitting a characteristic blue fluo-
lens rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as
X-Y
Base translation fluorescein to molecules that will specifically bind to certain
Tungsten mechanism
cellular components and thus allow the identification of these
halogen
lamp structures under the microscope (Figure 1–4b). Antibodies
labeled with fluorescent compounds are extremely important
Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing
mechanical components and the pathway of light from the Specific Molecules.)
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
Phase-Contrast Microscopy
■■ The condenser collects and focuses a cone of light that
illuminates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans-
■■ Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified
image of the object toward the eyepiece. Interchangeable light microscopes. Cellular detail is normally difficult to see
objectives with different magnifications routinely used in
in unstained tissues because all parts of the specimen have
histology include X4 for observing a large area (field) of the
tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros-
of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images
detailed areas. from transparent objects and, importantly, can be used with
■■ The two eyepieces or oculars magnify this image another living, cultured cells (Figure 1–5).
X10 and project it to the viewer, yielding a total magnifica-
Phase-contrast microscopy is based on the principle that
tion of X40, X100, or X400.
light changes its speed when passing through cellular and
(Used with permission from Nikon Instruments.) extracellular structures with different refractive indices. These
changes are used by the phase-contrast system to cause the
structures to appear lighter or darker in relation to each other.
stained tissue preparation to high-resolution digital images Because they allow the examination of cells without fixation or
and permits study of tissues using a computer or other digi- staining, phase-contrast microscopes are prominent tools in
tal device, without an actual stained slide or a microscope. In all cell culture laboratories. A modification of phase-contrast
this technique, regions of a glass-mounted specimen are cap- microscopy is differential interference contrast micros-
tured digitally in a grid-like pattern at multiple magnifications copy with Nomarski optics, which produces an image of liv-
using a specialized slide-scanning microscope and saved as ing cells with a more apparent three-dimensional (3D) aspect
thousands of consecutive image files. Software then converts (Figure 1–5c).
this dataset for storage on a server using a format that allows
access, visualization, and navigation of the original slide with
common web browsers or other devices. With advantages in Confocal Microscopy
cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light
light microscopes and collections of glass slides in histology is relatively large and fills the specimen. Stray (excess) light
laboratories for students. reduces contrast within the image and compromises the

01_Mescher_ch01_p001-016.indd 5 26/04/18 11:10 am

 6 CHAPTER 1 ■ Histology & Its Methods of Study
FIGURE 1–4 Appearance of cells with fluorescent microscopy.
N R
a b
Components of cells are often stained with compounds visible by
fluorescence microscopy.
(a) Acridine orange binds nucleic acids and causes DNA in cell
nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to
appear orange in these cells of a kidney tubule.
(b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole)
that binds DNA and with fluorescein phalloidin that binds actin
FIGURE 1–5 Unstained cells’ appearance in three types of light microscopy.
a b
Living neural crest cells growing in culture appear differently
with various techniques of light microscopy. Here the same field
of unstained cells, including two differentiating pigment cells, is
shown using three different methods (all X200):
(a) Bright-field microscopy: Without fixation and staining, only
the two pigment cells can be seen.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and
cytoplasmic structures with different refractive indices affect
01_Mescher_ch01_p001-016.indd 6
filaments show nuclei with blue fluorescence and actin filaments
stained green. Important information such as the greater density
of microfilaments at the cell periphery is readily apparent. (Both
X500)
(Figure 1–4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
Bloomington.)
c
in-phase light differently and produce an image of these features
in all the cells.
(c) Differential interference contrast microscopy: Cellular details
are highlighted in a different manner using Nomarski optics.
Phase-contrast microscopy, with or without differential interfer-
ence, is widely used to observe live cells grown in tissue culture.
(Used with permission from Dr Sherry Rogers, Department of Cell
Biology and Physiology, University of New Mexico, Albuquerque, NM.)
26/04/18 11:10 am
 Light Microscopy 7

such optical sections at a series of focal planes through the

FIGURE 1–6 Principle of confocal microscopy.
specimen allows them to be digitally reconstructed into a

C H A P T E R
3D image.

Laser Polarizing Microscopy

Polarizing microscopy allows the recognition of stained or
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits

1
Scanner vibrating in only one direction. If a second filter is placed in

Histology & Its Methods of Study ■ Light Microscopy

the microscope above the first one, with its main axis per-
Detector
pendicular to the first filter, no light passes through. If, how-
ever, tissue structures containing oriented macromolecules
are located between the two polarizing filters, their repeti-
tive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark
background (Figure 1–7). The ability to rotate the direction
Plate with of vibration of polarized light is called birefringence and is
pinhole
Beam
splitter FIGURE 1–7 Tissue appearance with bright-field
and polarizing microscopy.

Lens

Other
Focal plane out-of-focus
Specimen planes

Although a very small spot of light originating from one plane

of the section crosses the pinhole and reaches the detector,
rays originating from other planes are blocked by the blind.
Thus, only one very thin plane of the specimen is focused at a
time. The diagram shows the practical arrangement of a confo- a
cal microscope. Light from a laser source hits the specimen and
is reflected. A beam splitter directs the reflected light to a pin-
hole and a detector. Light from components of the specimen
that are above or below the focused plane is blocked by the
blind. The laser scans the specimen so that a larger area of the
specimen can be observed.

resolving power of the objective lens. Confocal microscopy

(Figure 1–6) avoids these problems and achieves high reso-
lution and sharp focus by using (1) a small point of high-
intensity light, often from a laser and (2) a plate with a
pinhole aperture in front of the image detector. The point
light source, the focal point of the lens, and the detector’s
pinpoint aperture are all optically conjugated or aligned to
each other in the focal plane (confocal), and unfocused light
does not pass through the pinhole. This greatly improves
resolution of the object in focus and allows the localization
of specimen components with much greater precision than
with the bright-field microscope.
Confocal microscopes include a computer-driven mirror
system (the beam splitter) to move the point of illumination
across the specimen automatically and rapidly. Digital images
captured at many individual spots in a very thin plane of focus
are used to produce an “optical section” of that plane. Creating
b
Polarizing light microscopy produces an image only of material
having repetitive, periodic macromolecular structure; features
without such structure are not seen. Pieces of thin, unsec-
tioned mesentery were stained with red picrosirius, orcein, and
hematoxylin, placed on slides and observed by bright-field (a)
and polarizing (b) microscopy.
(a) With bright-field microscopy, collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
(b) With polarizing microscopy, only the collagen fibers are
visible and these exhibit intense yellow or orange birefrin-
gence. (a: X40; b: X100)

01_Mescher_ch01_p001-016.indd 7 26/04/18 11:10 am

 8 CHAPTER 1 ■ Histology & Its Methods of Study

a feature of crystalline substances or substances containing
highly oriented molecules, such as cellulose, collagen, micro-
tubules, and actin filaments.
The utility of all light microscopic methods is greatly
extended through the use of digital cameras. Many features
of digitized histologic images can be analyzed quantitatively
using appropriate software. Such images can also be enhanced
to allow objects not directly visible through the eyepieces to be
examined on a monitor.
››ELECTRON MICROSCOPY
Transmission and scanning electron microscopes are based on
the interaction of tissue components with beams of electrons.
The wavelength in an electron beam is much shorter than that
of light, allowing a 1000-fold increase in resolution.
Transmission Electron Microscopy
The transmission electron microscope (TEM) is an imag-
ing system that permits resolution around 3 nm. This high
resolution allows isolated particles magnified as much as
400,000 times to be viewed in detail. Very thin (40-90 nm),
resin-embedded tissue sections are typically studied by TEM
at magnifications up to approximately 120,000 times.
Figure 1–8a indicates the components of a TEM and the
basic principles of its operation: a beam of electrons focused
using electromagnetic “lenses” passes through the tissue sec-
tion to produce an image with black, white, and intermediate

FIGURE 1–8 Electron microscopes.

Electron gun Electron gun

Cathode Cathode

3 mm
Anode Anode

Copper grid
Condensor lens with three sections Lens

Specimen Column
Objective lens holder Lens
Column Scanner
Intermediate lens Electron detector

TEM image Lens SEM image

Projector lens

Image on viewing
screen Specimen
Electron detector
with CCD camera

(a) Transmission electron microscope (b) Scanning electron microscope

Electron microscopes are large instruments generally housed in a
specialized EM facility.
(a) Schematic view of the major components of a transmission elec-
tron microscope (TEM), which is configured rather like an upside-
down light microscope. With the microscope column in a vacuum, a
metallic (usually tungsten) filament (cathode) at the top emits elec-
trons that travel to an anode with an accelerating voltage between
60 and 120 kV. Electrons passing through a hole in the anode form
a beam that is focused electromagnetically by circular electric
coils in a manner analogous to the effect of optical lenses on light.
The first lens is a condenser focusing the beam on the section.
Some electrons interact with atoms in the section, being absorbed
or scattered to different extents, while others are simply transmit-
ted through the specimen with no interaction. Electrons reaching
the objective lens form an image that is then magnified and finally
projected on a fluorescent screen or a charge-coupled device
(CCD) monitor and camera.
In a TEM image areas of the specimen through which electrons
passed appear bright (electron lucent), while denser areas or
those that bind heavy metal ions during specimen preparation
absorb or deflect electrons and appear darker (electron dense).
Such images are therefore always black, white, and shades of gray.
(b) The scanning electron microscope (SEM) has many similarities
to a TEM. However, here the focused electron beam does not pass
through the specimen, but rather is moved sequentially (scanned)
from point to point across its surface similar to the way an electron
beam is scanned across a television tube or screen. For SEM speci-
mens are coated with metal atoms with which the electron beam
interacts, producing reflected electrons and newly emitted secondary
electrons. All of these are captured by a detector and transmitted to
amplifiers and processed to produce a black-and-white image on the
monitor. The SEM shows only surface views of the coated specimen
but with a striking 3D, shadowed quality. The inside of organs or cells
can be analyzed after sectioning to expose their internal surfaces.

01_Mescher_ch01_p001-016.indd 8 26/04/18 11:10 am


shades of gray regions. These regions of an electron micro-
graph correspond to tissue areas through which electrons
passed readily (appearing brighter or electron-lucent) and
areas where electrons were absorbed or deflected (appearing
darker or more electron-dense). To improve contrast and reso-
lution in TEM, compounds with heavy metal ions are often
added to the fixative or dehydrating solutions used for tissue
preparation. These include osmium tetroxide, lead citrate,
and uranyl compounds, which bind cellular macromolecules,
increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that
allow TEM study of cells without fixation or embedding and
have been particularly useful in the study of membrane struc-
ture. In these methods, very small tissue specimens are rap-
idly frozen in liquid nitrogen and then cut or fractured with a
knife. A replica of the frozen exposed surface is produced in a
vacuum by applying thin coats of vaporized platinum or other
metal atoms. After removal of the organic material, the replica
of the cut surface can be examined by TEM. With membranes
the random fracture planes often split the lipid bilayers, expos-
ing protein components whose size, shape, and distribution
are difficult to study by other methods.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) provides a high-
resolution view of the surfaces of cells, tissues, and organs. Like
the TEM, this microscope produces and focuses a very narrow
beam of electrons, but in this instrument the beam does not
pass through the specimen (Figure 1–8b). Instead, the surface
of the specimen is first dried and spray-coated with a very thin
FIGURE 1–9 Microscopic autoradiography.
a b
Autoradiographs are tissue preparations in which particles called
silver grains indicate the cells or regions of cells in which specific
macromolecules were synthesized just prior to fixation. Shown
here are autoradiographs from the salivary gland of a mouse
injected with 3H-fucose 8 hours before tissue fixation. Fucose was
incorporated into oligosaccharides, and the free 3H-fucose was
removed during fixation and sectioning of the gland. Autoradio-
graphic processing and microscopy reveal locations of newly syn-
thesized glycoproteins containing that sugar.
01_Mescher_ch01_p001-016.indd 9
Autoradiography 9
layer of heavy metal (often gold) that reflects electrons in a
beam scanning the specimen. The reflected electrons are cap-
C H A P T E R
tured by a detector, producing signals that are processed to pro-
duce a black-and-white image. SEM images are usually easy to
interpret because they present a three-dimensional view that
appears to be illuminated in the same way that large objects are
seen with highlights and shadows caused by light.
1
››AUTORADIOGRAPHY
Histology & Its Methods of Study ■ Autoradiography
Microscopic autoradiography is a method of localizing
newly synthesized macromolecules in cells or tissue sections.
Radioactively labeled metabolites (nucleotides, amino acids,
sugars) provided to the living cells are incorporated into spe-
cific macromolecules (DNA, RNA, protein, glycoproteins, and
polysaccharides) and emit weak radiation that is restricted
to those regions where the molecules are located. Slides with
radiolabeled cells or tissue sections are coated in a darkroom
with photographic emulsion in which silver bromide crystals
act as microdetectors of the radiation in the same way that
they respond to light in photographic film. After an adequate
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia-
tion produce small black grains of metallic silver, which under
either the light microscope or TEM indicate the locations of
radiolabeled macromolecules in the tissue (Figure 1–9).
Much histological information becomes available by
autoradiography. If a radioactive precursor of DNA (such
as tritium-labeled thymidine) is used, it is possible to know
which cells in a tissue (and how many) are replicating DNA
G
G
(a) Black grains of silver from the light-sensitive material coating
the specimen are visible over cell regions with secretory granules
and the duct indicating glycoprotein locations. (X1500)
(b) The same tissue prepared for TEM autoradiography shows sil-
ver grains with a coiled or amorphous appearance again localized
mainly over the granules (G) and in the gland lumen (L). (X7500)
(Figure 1–9b, used with permission from Drs Ticiano G. Lima and
A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
26/04/18 11:10 am
 10 CHAPTER 1 ■ Histology & Its Methods of Study
and preparing to divide. Dynamic events may also be analyzed.
For example, if one wishes to know where in the cell protein is
produced, if it is secreted, and its path in the cell before being
secreted, several animals are injected with a radioactive amino
acid and tissues collected at different times after the injections.
Autoradiography of the tissues from the sequential times will
indicate the migration of the radioactive proteins.
››CELL & TISSUE CULTURE
Live cells and tissues can be maintained and studied outside
the body in culture (in vitro). In the organism (in vivo), cells
are bathed in fluid derived from blood plasma and containing
many different molecules required for survival and growth.
Cell culture allows the direct observation of cellular behavior
under a phase-contrast microscope, and many experiments
technically impossible to perform in the intact animal can be
accomplished in vitro.
The cells and tissues are grown in complex solutions of
known composition (salts, amino acids, vitamins) to which
serum or specific growth factors are added. Cells to be cultured
are dispersed mechanically or enzymatically from a tissue or
organ and placed with sterile procedures in a clear dish to
which they adhere, usually as a single layer (see Figure 1–5).
Such preparations are called primary cell cultures. Some
cells can be maintained in vitro for long periods because they
become immortalized and constitute a permanent cell line.
Most cells obtained from normal tissues have a finite, geneti-
cally programmed life span. However, certain changes (some
related to oncogenes; see Chapter 3) can promote cell immor-
tality, a process called transformation, and are similar to
the initial changes in a normal cell’s becoming a cancer cell.
Improvements in culture technology and use of specific growth
factors now allow most cell types to be maintained in vitro.
As shown in Chapter 2, incubation of living cells in vitro
with a variety of new fluorescent compounds that are seques-
tered and metabolized in specific compartments of the cell
provides a new approach to understanding these compart-
ments both structurally and physiologically. Other histologic
techniques applied to cultured cells have been particularly
important for understanding the locations and functions of
microtubules, microfilaments, and other components of the
cytoskeleton.
› ›› MEDICAL APPLICATION
Cell culture is very widely used to study molecular changes
that occur in cancer; to analyze infectious viruses, myco-
plasma, and some protozoa; and for many routine genetic or
chromosomal analyses. Cervical cancer cells from a patient
later identified as Henrietta Lacks, who died from the disease
in 1951, were used to establish one of the first cell lines,
called HeLa cells, which are still used in research on cellular
structure and function throughout the world.
01_Mescher_ch01_p001-016.indd 10
››ENZYME HISTOCHEMISTRY
Enzyme histochemistry (or cytochemistry) is a method for
localizing cellular structures using a specific enzymatic activ-
ity present in those structures. To preserve the endogenous
enzymes, histochemical procedures usually use unfixed or
mildly fixed tissue, which is sectioned on a cryostat to avoid
adverse effects of heat and organic solvents on enzymatic
activity. For enzyme histochemistry: (1) tissue sections are
immersed in a solution containing the substrate of the enzyme
to be localized; (2) the enzyme is allowed to act on its sub-
strate; (3) the section is then put in contact with a marker
compound that reacts with a product of the enzymatic action
on the substrate; and (4) the final product from the marker,
which must be insoluble and visible by light or electron
microscopy, precipitates over the site of the enzymes, identify-
ing their location.
Examples of enzymes that can be detected histochemi-
cally include the following:
■■ Phosphatases, which remove phosphate groups from
macromolecules (Figure 1–10).
■■ Dehydrogenases, which transfer hydrogen ions from
one substrate to another, such as many enzymes of the
citric acid (Krebs) cycle, allowing histochemical identifi-
cation of such enzymes in mitochondria.
■■ Peroxidase, which promotes the oxidation of sub-
strates with the transfer of hydrogen ions to hydrogen
peroxide.
› ›› MEDICAL APPLICATION
Many enzyme histochemical procedures are used in the
medical laboratory, including Perls’ Prussian blue reaction for
iron (used to diagnose the iron storage diseases, hemochro-
matosis and hemosiderosis), the PAS-amylase and alcian blue
reactions for polysaccharides (to detect glycogenosis and
mucopolysaccharidosis), and reactions for lipids and sphin-
golipids (to detect sphingolipidosis).
››VISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also
be identified by using tagged compounds or macromolecules
that bind specifically with the molecule of interest. The com-
pounds that interact with the molecule must be visible with
the light or electron microscope, often by being tagged with a
detectible label. The most commonly used labels are fluores-
cent compounds, radioactive atoms that can be detected with
autoradiography, molecules of peroxidase or other enzymes
that can be detected with histochemistry, and metal (usually
gold) particles that can be seen with light and electron micros-
copy. These methods can be used to detect and localize specific
sugars, proteins, and nucleic acids.
26/04/18 11:10 am

FIGURE 1–10 Enzyme histochemistry.
L ■■
L L
aa
Ly
Ly
b N
(a) Micrograph of cross sections of kidney tubules treated
histochemically to demonstrate alkaline phosphatases (with
maximum activity at an alkaline pH) showing strong activity of
this enzyme at the apical surfaces of the cells at the lumens (L)
of the tubules. (X200)
(b) TEM image of a kidney cell in which acid phosphatase has
been localized histochemically in three lysosomes (Ly) near the
nucleus (N). The dark material within these structures is lead
phosphate that precipitated in places with acid phosphatase
activity. (X25,000)
(Figure 1–10b, used with permission from Dr Eduardo
Katchburian, Department of Morphology, Federal University of
São Paulo, Brazil.)
01_Mescher_ch01_p001-016.indd 11
Visualizing Specific Molecules 11
Examples of molecules that interact specifically with
other molecules include the following:
C H A P T E R
■■ Phalloidin, a compound extracted from mushroom,
Amanita phalloides, interacts strongly with the actin pro-
tein of microfilaments.
Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fc region of antibody molecules, and
can therefore be used to localize naturally occurring or
1
applied antibodies bound to cell structures.
Histology & Its Methods of Study ■ Visualizing Specific Molecules
■■ Lectins, glycoproteins derived mainly from plant seeds,
bind to carbohydrates with high affinity and specificity.
Different lectins bind to specific sugars or sequences of
sugar residues, allowing fluorescently labeled lectins to
be used to stain specific glycoproteins or other macro-
molecules bearing specific sequences of sugar residues.
Immunohistochemistry
A highly specific interaction between macromolecules is that
between an antigen and its antibody. For this reason, labeled
antibodies are routinely used in immunohistochemistry
to identify and localize many specific proteins, not just
those with enzymatic activity that can be demonstrated by
histochemistry.
The body’s immune cells interact with and produce anti-
bodies against other macromolecules—called antigens—that
are recognized as “foreign,” not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
noglobulin family of glycoproteins and are secreted by lym-
phocytes. These molecules normally bind specifically to their
provoking antigens and help eliminate them.
Widely applied for both research and diagnostic pur-
poses, every immunohistochemical technique requires an
antibody against the protein that is to be detected. This means
that the protein must have been previously purified using bio-
chemical or molecular methods so that antibodies against it
can be produced. To produce antibodies against protein x of a
certain animal species (eg, a human or rat), the isolated pro-
tein is injected into an animal of another species (eg, a rabbit
or a goat). If the protein’s amino acid sequence is sufficiently
different for this animal to recognize it as foreign—that is, as
an antigen—the animal will produce antibodies against the
protein.
Different groups (clones) of lymphocytes in the injected
animal recognize different parts of protein x and each clone
produces an antibody against that part. These antibodies are
collected from the animal’s plasma and constitute a mixture
of polyclonal antibodies, each capable of binding a different
region of protein x.
It is also possible, however, to inject protein x into a
mouse and a few days later isolate the activated lymphocytes
and place them into culture. Growth and activity of these cells
can be prolonged indefinitely by fusing them with lymphocytic
tumor cells to produce hybridoma cells. Different hybridoma
clones produce different antibodies against the several parts of
26/04/18 11:10 am
 12 CHAPTER 1 ■ Histology & Its Methods of Study
protein x, and each clone can be isolated and cultured sepa-
rately so that the different antibodies against protein x can be
collected separately. Each of these antibodies is a monoclo-
nal antibody. An advantage to using a monoclonal antibody
rather than polyclonal antibodies is that it can be selected to
be highly specific and to bind strongly to the protein to be
detected, with less nonspecific binding to other proteins that
are similar to the one of interest.
In immunohistochemistry, a tissue section that one
believes contains the protein of interest is incubated in a solu-
tion containing antibody (either monoclonal or polyclonal)
against this protein. The antibody binds specifically to the
protein and after a rinse the protein’s location in the tissue or
cells can be seen with either the light or electron microscope
by visualizing the antibody. Antibodies are commonly tagged
with fluorescent compounds, with peroxidase or alkaline
phosphatase for histochemical detection, or with electron-
dense gold particles for TEM.
As Figure 1–11 indicates, there are direct and indirect
methods of immunocytochemistry. The direct method just
involves a labeled antibody that binds the protein of interest.
Indirect immunohistochemistry involves sequential
application of two antibodies and additional washing steps. The
(primary) antibody specifically binding the protein of interest
is not labeled. The detectible tag is conjugated to a second-
ary antibody made in an animal species different (“foreign”)
from that which made the primary antibody. For example, pri-
mary antibodies made by mouse lymphocytes (such as most
monoclonal antibodies) are specifically recognized and bound
by antibodies made in a rabbit or goat injected with mouse
antibody immunoglobulin.
FIGURE 1–11 Immunocytochemistry techniques.
Labeled Unlabeled
antibody primary
antibody
Antigen Antigen
Tissue section
Glass slide
Direct
Immunocytochemistry (or immunohistochemistry) can be direct
or indirect. Direct immunocytochemistry (left) uses an antibody
made against the tissue protein of interest and tagged directly
with a label such as a fluorescent compound or peroxidase. When
placed with the tissue section on a slide, these labeled antibod-
ies bind specifically to the protein (antigen) against which they
were produced and can be visualized by the appropriate method.
Indirect immunocytochemistry (right) uses first a primary
antibody made against the protein (antigen) of interest and
applied to the tissue section to bind its specific antigen. Then a
01_Mescher_ch01_p001-016.indd 12
The indirect method is used more widely in research and
pathologic tests because it is more sensitive, with the extra
level of antibody binding serving to amplify the visible signal.
Moreover, the same preparation of labeled secondary antibody
can be used in studies with different primary antibodies (spe-
cific for different antigens) as long as all these are made in the
same species. There are other indirect methods that involve the
use of other intermediate molecules, such as the biotin-avidin
technique, which are also used to amplify detection signals.
Examples of indirect immunocytochemistry are shown in
Figure 1–12, demonstrating the use of this method with cells
in culture or after tissue sectioning for both light microscopy
and TEM.
› ›› MEDICAL APPLICATION
Because cells in some diseases, including many cancer cells,
often produce proteins unique to their pathologic condition,
immunohistochemistry can be used by pathologists to diag-
nose many diseases, including certain types of tumors and
some virus-infected cells. Table 1–1 shows some applications
of immunocytochemistry routinely used in clinical practice.
Hybridization Techniques
Hybridization usually implies the specific binding between
two single strands of nucleic acid, which occurs under appro-
priate conditions if the strands are complementary. The greater
the similarities of their nucleotide sequences, the more read-
ily the complementary strands form “hybrid” double-strand
molecules. Hybridization at stringent conditions allows the
Labeled
secondary
antibody
Indirect
labeled secondary antibody is obtained that was (1) made in
another species against immunoglobulin proteins (antibodies)
from the species in which the primary antibodies were made and
(2) labeled with a fluorescent compound or peroxidase. When
the labeled secondary antibody is applied to the tissue section, it
specifically binds the primary antibodies, indirectly labeling the
protein of interest on the slide. Because more than one labeled
secondary antibody can bind each primary antibody molecule,
labeling of the protein of interest is amplified by the indirect
method.
26/04/18 11:10 am
 Visualizing Specific Molecules 13

FIGURE 1–12 Cells and tissues stained by immunohistochemistry.

1 C H A P T E R
Histology & Its Methods of Study ■ Visualizing Specific Molecules
c
a

the cytoplasm. Primary antibodies against the filament pro-

b
Immunocytochemical methods to localize specific proteins can
be applied to either light microscopic or TEM preparations using
a variety of labels.
(a) A single cultured uterine cell stained fluorescently to reveal
a meshwork of intermediate filaments (green) throughout
tein desmin and fluorescein isothiocyanate (FITC)-labeled
secondary antibodies were used in the indirect staining
technique, with the nucleus counterstained blue with DAPI.
(X650)
(b) A section of small intestine treated with an antibody against
the enzyme lysozyme. The secondary antibody labeled with
peroxidase was then applied and the localized brown color
produced histochemically with the peroxidase substrate
3,3′-diamino-azobenzidine (DAB). The method demonstrates
lysozyme-containing structures in scattered macrophages and
in the large clusters of cells. Nuclei were counterstained with
hematoxylin. (X100)
(c) A section of pancreatic cells in a TEM preparation incubated
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin-
ity toward antibody molecules and the resulting image reveals
the presence of amylase with the gold particles localized as very
small black dots over dense secretory granules and developing
granules (left). With specificity for immunoglobulin molecules,
labeled protein A can be used to localize any primary antibody.
(X5000)
(Figure 1–12c, used with permission from Dr Moise Bendayan,
Departments of Pathology and Cell Biology, University of Montreal,
Montreal, Canada.)

TABLE 1–1    Examples of specific antigens with diagnostic importance.

Antigens Diagnosis

Specific cytokeratins Tumors of epithelial origin

Protein and polypeptide hormones Certain endocrine tumors
Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast
Steroid hormone receptors Breast duct cell tumors
Antigens produced by viruses Specific virus infections

01_Mescher_ch01_p001-016.indd 13 26/04/18 11:10 am

 14 CHAPTER 1 ■ Histology & Its Methods of Study
specific identification of sequences in genes or RNA. This can
occur with cellular DNA or RNA when nucleic acid sequences
in solution are applied directly to prepared cells and tissue sec-
tions, a procedure called in situ hybridization (ISH).
This technique is ideal for (1) determining if a cell has
a specific sequence of DNA, such as a gene or part of a gene
(Figure 1–13), (2) identifying the cells containing specific mes-
senger RNAs (mRNAs) (in which the corresponding gene is
being transcribed), or (3) determining the localization of a gene
in a specific chromosome. DNA and RNA of the cells must be
initially denatured by heat or other agents to become completely
single-stranded, and the nucleotide sequences of interest are
detected with probes consisting of single-stranded comple-
mentary DNA (cDNA). The probe may be obtained by cloning,
by polymerase chain reaction (PCR) amplification of the target
sequence, or by chemical synthesis if the desired sequence is
short. The probe is tagged with nucleotides containing a radio-
active isotope (localized by autoradiography) or modified with
a small compound such as digoxigenin (identified by immuno-
cytochemistry). A solution containing the probe is placed over
the specimen under conditions allowing hybridization and after
the excess unbound probe is washed off, the localization of the
hybridized probe is revealed through its label.
FIGURE 1–13 In situ hybridization (ISH).
In situ hybridization of this tissue section with probes for the
human papillomavirus (HPV) reveals the presence of many
cells containing the virus. The section was incubated with a
solution containing a digoxigenin-labeled complementary
DNA (cDNA) probe for the HPV DNA. The probe was then
visualized by direct immunohistochemistry using peroxidase-
labeled antibodies against digoxigenin. This procedure stains
brown only those cells containing HPV. (X400; H&E)
(Used with permission from Dr Jose E. Levi, Virology Lab, Insti-
tute of Tropical Medicine, University of São Paulo, Brazil.)
01_Mescher_ch01_p001-016.indd 14
› ›› MEDICAL APPLICATION
Warts on the skin of the genitals and elsewhere are due to
infection with the human papillomavirus (HPV) which causes
the characteristic benign proliferative growth. As shown in
Figure 1–12, such virus-infected cells can often be demon-
strated by ISH. Certain cancer cells with unique or elevated
expression of specific genes are also localized in tumors and
studied microscopically by ISH.
››INTERPRETATION OF STRUCTURES
IN TISSUE SECTIONS
In studying and interpreting stained tissue sections, it is
important to remember that microscopic preparations are the
end result of a series of processes that began with collecting
the tissue and ended with mounting a coverslip on the slide.
Certain steps in this procedure may distort the tissues slightly,
producing minor structural abnormalities called artifacts not
present in the living tissue.
One such distortion is minor shrinkage of cells or tissue
regions produced by the fixative, by the ethanol, or by the heat
needed for paraffin embedding. Shrinkage can create artificial
spaces between cells and other tissue components. Such spaces
can also result from the loss of lipids or low-molecular-weight
substances not preserved by the fixative or removed by the
dehydrating and clearing fluids. Slight cracks in sections may
also appear as large spaces in the tissue.
Other artifacts may include small wrinkles in the section
(which the novice may confuse with linear structures in tissue)
and precipitates from the stain (which may be confused with
cellular structures such as cytoplasmic granules). Students must
be aware of the existence of artifacts and able to recognize them.
Another difficulty in the study of histologic sections is the
impossibility of differentially staining all tissue components on
one slide. A single stain can seldom demonstrate well nuclei,
mitochondria, lysosomes, basement membranes, elastic fibers,
etc. With the light microscope, it is necessary to examine prepa-
rations stained by different methods before an idea of the whole
composition and structure of a cell or tissue can be obtained. The
TEM allows the observation of cells with all its internal structures
and surrounding ECM components, but only a few cells in a tis-
sue can be conveniently studied in these very small samples.
Finally, when a structure’s three-dimensional volume is
cut into very thin sections, the sections appear microscopically
to have only two dimensions: length and width. When examin-
ing a section under the microscope, the viewer must always keep
in mind that components are missing in front of and behind
what is being seen because many tissue structures are thicker
than the section. Round structures seen microscopically may
actually be portions of spheres or tubes. Because structures in
a tissue have different orientations, their two-dimensional (2D)
appearance will also vary depending on the plane of section. A
single convoluted tube will appear in a tissue section as many
separate rounded or oval structures (Figure 1–14).
26/04/18 11:10 am
 Interpretation of Structures in Tissue Sections 15

FIGURE 1–14 Interpretation of 3D structures in 2D sections.

1 C H A P T E R
Histology & Its Methods of Study ■ Interpretation of Structures in Tissue Sections
In thin sections 3D structures appear to have only two dimensions.
Such images must be interpreted correctly to understand the actual
structure of tissue and organ components. For example, blood ves-
sels and other tubular structures appear in sections as round or oval
shapes whose size and shape depend on the transverse or oblique
angle of the cut. A highly coiled tube will appear as several round
and oval structures. In TEM sections of cells, round structures may
represent spherical organelles or transverse cuts through tubular
organelles such as mitochondria. It is important to develop such
interpretive skill to understand tissue and cell morphology in micro-
scopic preparations.

Histology & Its Methods of Study SUMMARY OF KEY POINTS

Preparation of Tissues for Study Autoradiography

■■ Chemical fixatives such as formalin are used to preserve tissue
structure by cross-linking and denaturing proteins, inactivating
enzymes, and preventing cell autolysis or self-digestion.
■■ Dehydration of the fixed tissue in alcohol and clearing in organic
solvents prepare it for embedding and sectioning.
■■ Embedding in paraffin wax or epoxy resin allows the tissue to be
cut into very thin sections (slices) with a microtome.
■■ Sections are mounted on glass slides for staining, which is
required to reveal specific cellular and tissue components with the
microscope.
■■ The most commonly used staining method is a combination of the
stains H&E, which act as basic and acidic dyes, respectively.
■■ Cell substances with a net negative (anionic) charge, such as DNA
and RNA, react strongly with hematoxylin and basic stains; such
material is said to be “basophilic.”
■■ Cationic substances, such as collagen and many cytoplasmic pro-
teins react with eosin and other acidic stains and are said to be
“acidophilic.”
Light Microscopy
■■ Bright-field microscopy, the method most commonly used by
both students and pathologists, uses ordinary light and the colors
are imparted by tissue staining.
■■ Fluorescence microscopy uses UV light, under which only fluo-
rescent molecules are visible, allowing localization of fluorescent
probes which can be much more specific than routine stains.
■■ Phase-contrast microscopy uses the differences in refractive
index of various natural cell and tissue components to produce an
image without staining, allowing observation of living cells.
■■ Confocal microscopy involves scanning the specimen at succes-
sive focal planes with a focused light beam, often from a laser, and
produces a 3D reconstruction from the images.
■■ This process localizes cell components synthesized using radioactive
precursors by detecting silver grains produced by weakly emitted radia-
tion in a photographic emulsion coating the tissue section or cells.
■■ With either light microscopy or TEM, autoradiography permits
unique studies of processes such as tissue growth (using radioactive
DNA precursors) or cellular pathways of macromolecular synthesis.
Cell & Tissue Culture
■■ Cells can be grown in vitro from newly explanted tissues (primary
cultures) or as long-established cell lines and can be examined in the
living state by phase-contrast light microscopy.
Enzyme Histochemistry
■■ Histochemical (or cytochemical) techniques use specific enzy-
matic activities in lightly fixed or unfixed tissue sections to produce
visible products in the specific enzyme locations.
■■ Fixation and paraffin embedding denatures most enzymes, so histo-
chemistry usually uses frozen tissue sectioned with a cryostat.
■■ Enzyme classes for which histochemical study is useful include
phosphatases, dehydrogenases, and peroxidases, with peroxidase
often conjugated to antibodies used in immunohistochemistry.
Visualizing Specific Molecules
■■ Some substances specifically bind certain targets in cells.
■■ Immunohistochemistry is based on specific reactions between an anti-
gen and antibodies labeled with visible markers, often fluorescent com-
pounds or peroxidase for light microscopy and gold particles for TEM.
■■ If the cell or tissue antigen of interest is detected by directly binding
a labeled primary antibody specific for that antigen, the process is
considered direct immunohistochemistry.
■■ Indirect immunohistochemistry uses an unlabeled primary anti-
body that is detected bound to its antigen with labeled secondary
antibodies.

01_Mescher_ch01_p001-016.indd 15 26/04/18 11:10 am

 16 CHAPTER 1 ■ Histology & Its Methods of Study

■■ The indirect immunohistochemical method is more commonly used
because the added level of antibody binding amplifies the signal
detected and provides greater technical flexibility.
■■ Specific gene sequences or mRNAs of cells can be detected micro-
scopically using labeled cDNA probes in a technique called in situ
hybridization (ISH).
Interpretation of Structures in Tissue Sections
■■ Many steps in tissue processing, slide preparation, and staining can
introduce minor artifacts such as spaces and precipitates that are not
normally present in the living tissue and must be recognized.
■■ Sections of cells or tissues are essentially 2D planes through 3D
structures, and understanding this fact is important for their correct
interpretation and study.

Histology & Its Methods of Study ASSESS YOUR KNOWLEDGE

1. In preparing tissue for routine light microscopic study, which 7. Microscopic autoradiography uses radioactivity and can be
procedure immediately precedes clearing the specimen with an employed to study what features in a tissue section?
organic solvent? a. The types of enzymes found in various cell locations
a. Dehydration b. Cellular sites where various macromolecules are synthesized
b. Fixation c. The sequences of mRNA made in the cells
c. Staining d. The dimensions of structures within the cells
d. Clearing e. The locations of genes transcribed for specific mRNA
e. Embedding
8. To identify and localize a specific protein within cells or the ECM,
2. Which of the following staining procedures relies on the cationic one would best use what approach?
and anionic properties of the material to be stained? a. Autoradiography
a. Enzyme histochemistry b. Enzyme histochemistry
b. PAS reaction c. Immunohistochemistry
c. H&E staining d. TEM
d. Immunohistochemistry e. Polarizing microscopy
e. Metal impregnation techniques
9. In situ hybridization is a histologic technique used to visualize what
3. In a light microscope used for histology, resolution and magnifica- type of macromolecule?
tion of cells are largely dependent on which component? a. Proteins
a. Condenser b. Carbohydrates
b. Objective lens c. Certain enzymes
c. Eyepieces or ocular lenses d. Nucleic acids
d. Specimen slide e. Lipids
e. The control for illumination intensity
10. Hospital laboratories frequently use unfixed, frozen tissue specimens
4. Cellular storage deposits of glycogen, a free polysaccharide, could sectioned with a cryostat for rapid staining, microscopic examina-
best be detected histologically using what procedure? tion, and diagnosis of pathologic conditions. Besides saving much
a. Autoradiography time by avoiding fixation and procedures required for paraffin
b. Electron microscopy embedding, frozen sections retain and allow study of what macro-
c. Enzyme histochemistry molecules normally lost in the paraffin procedure?
d. H&E staining a. Carbohydrates
e. PAS reaction b. Small mRNA
c. Basic proteins
5. Adding heavy metal compounds to the fixative and ultrathin sec- d. Acidic proteins
tioning of the embedded tissue with a glass knife are techniques e. Lipids
used for which histologic procedure?
a. Scanning electron microscopy
b. Fluorescent microscopy
c. Enzyme histochemistry
d. Confocal microscopy
e. TEM
6. Resolution in electron microscopy greatly exceeds that of light
microscopy due to which of the following?
a. The wavelength of the electrons in the microscope beam is
shorter than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved
quality.
c. For electron microscopy the tissue specimen does not require
staining.
d. The electron microscope allows much greater magnification of
a projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled Answers: 1a, 2c, 3b, 4e, 5e, 6a, 7b, 8c, 9d, 10e
than a light microscope.

01_Mescher_ch01_p001-016.indd 16 26/04/18 11:10 am

 C H A P T E R
CELL DIFFERENTIATION
2 The Cytoplasm
17
THE PLASMA MEMBRANE 17
Transmembrane Proteins & Membrane Transport 19
Transport by Vesicles: Endocytosis & Exocytosis 21
Signal Reception & Transduction 23
CYTOPLASMIC ORGANELLES 27
Ribosomes27
Endoplasmic Reticulum 28
Golgi Apparatus 31
Secretory Granules 33
Lysosomes34
C ells and extracellular material together comprise the
tissues that make up animal organs. In all tissues,
cells are the basic structural and functional units, the
smallest living parts of the body. Animal cells are enclosed
by cell membranes and are eukaryotic, each with a distinct,
membrane-enclosed nucleus surrounded by cytoplasm, fluid
containing a system of membranous organelles, nonmembra-
nous molecular assemblies, and a cytoskeleton. In contrast,
the smaller prokaryotic cells of bacteria typically have a cell
wall and lack nuclei and membranous cytoplasmic structures.
››CELL DIFFERENTIATION
The average adult human body consists of nearly 40 trillion
cells, according to the best available estimate. These cells exist
as hundreds of histologically distinct cell types, all derived
from the zygote, and the single cell formed by the merger of a
spermatozoon with an oocyte at fertilization. The first zygotic
cellular divisions produce cells called blastomeres, and as
part of the early embryo’s inner cell mass blastomeres give
rise to all tissue types of the fetus. Explanted to tissue culture
cells of the inner cell mass are called embryonic stem cells.
Most cells of the fetus undergo a specialization process called
differentiation in which they predominantly express sets of
genes that mediate specific cytoplasmic activities, becoming
efficiently organized in tissues with specialized functions and
usually changing their shape accordingly. For example, muscle
cell precursors elongate into long, fiber-like cells containing
large arrays of actin and myosin. All animal cells contain actin
filaments and myosins, but muscle cells are specialized for
02_Mescher_ch02_p017-052.indd 17
Proteasomes37
Mitochondria38
Peroxisomes39
THE CYTOSKELETON 42
Microtubules43
Microfilaments (Actin Filaments) 44
Intermediate Filaments 45
INCLUSIONS47
SUMMARY OF KEY POINTS 51
ASSESS YOUR KNOWLEDGE 52
using these proteins to convert chemical energy into forceful
contractions.
Major cellular functions performed by specialized cells in the
body are listed in Table 2–1. It is important to understand that the
functions listed there can be performed by most cells of the body;
specialized cells have greatly expanded their capacity for one or
more of these functions during differentiation. Changes in cells’
microenvironments under normal and pathologic conditions
can cause the same cell type to have variable features and activi-
ties. Cells that appear similar structurally often have different
families of receptors for signaling molecules such as hormones
and extracellular matrix (ECM) components, causing them to
behave differently. For example, because of their diverse arrays of
receptors, breast fibroblasts and uterine smooth muscle cells are
exceptionally sensitive to female sex hormones, while most other
fibroblasts and smooth muscle cells are insensitive.
››THE PLASMA MEMBRANE
The plasma membrane (cell membrane or plasmalemma)
that envelops every eukaryotic cell consists of phospholipids,
cholesterol, and proteins, with oligosaccharide chains covalently
linked to many of the phospholipids and proteins. This limiting
membrane functions as a selective barrier regulating the passage
of materials into and out of the cell and facilitating the transport
of specific molecules. One important role of the cell membrane is
to keep constant the ion content of cytoplasm, which differs from
that of the extracellular fluid. Membrane proteins also perform a
number of specific recognition and signaling functions, playing a
key role in the interactions of the cell with its environment.
17
27/04/18 6:48 pm
 18 CHAPTER 2 ■ The Cytoplasm

abundant in the outer half, while phosphatidylserine and

       
Differentiated cells typically
TABLE 2–1 specialize in one activity.
phosphatidylethanolamine are more concentrated in the inner
layer. Some of the outer layer’s lipids, known as glycolipids,
General Cellular Activity Specialized Cell(s) include oligosaccharide chains that extend outward from the
cell surface and contribute to a delicate cell surface coating
Movement Muscle and other contractile called the glycocalyx (Figures 2–1b and 2–2). With the trans-
cells mission electron microscope (TEM) the cell membrane—as
Form adhesive and tight Epithelial cells well as all cytoplasmic membranes—may exhibit a trilaminar
junctions between cells appearance after fixation in osmium tetroxide; osmium binds
Synthesize and secrete Fibroblasts, cells of bone and the polar heads of the phospholipids and the oligosaccharide
components of the extracellular cartilage chains, producing the two dark outer lines that enclose the
matrix light band of osmium-free fatty acids (Figure 2–1b).
Convert physical and chemical Neurons and sensory cells Proteins are major constituents of membranes (~50%
stimuli into action potentials by weight in the plasma membrane). Integral proteins are
Synthesis and secretion of Cells of digestive glands incorporated directly within the lipid bilayer, whereas periph-
degradative enzymes eral proteins are bound to one of the two membrane surfaces,
Synthesis and secretion of Cells of mucous glands
particularly on the cytoplasmic side (Figure 2–2). Peripheral
glycoproteins proteins can be extracted from cell membranes with salt solu-
tions, whereas integral proteins can be extracted only by using
Synthesis and secretion of Certain cells of the adrenal
steroids gland, testis, and ovary
detergents to disrupt the lipids. The polypeptide chains of
many integral proteins span the membrane, from one side to
Ion transport Cells of the kidney and the other, several times and are accordingly called multipass
salivary gland ducts
proteins. Integration of the proteins within the lipid bilayer
Intracellular digestion Macrophages and neutrophils is mainly the result of hydrophobic interactions between the
Lipid storage Fat cells lipids and nonpolar amino acids of the proteins.
Freeze-fracture electron-microscope studies of mem-
Metabolite absorption Cells lining the intestine
branes show that parts of many integral proteins protrude
from both the outer or inner membrane surface (Figure 2–2b).
Although the plasma membrane defines the outer limit of Like those of glycolipids, the carbohydrate moieties of glyco-
the cell, a continuum exists between the interior of the cell and proteins project from the external surface of the plasma mem-
extracellular macromolecules. Certain plasma membrane pro- brane and contribute to the glycocalyx (Figure 2–3). They are
teins, the integrins, are linked to both the cytoskeleton and important components of proteins acting as receptors, which
ECM components and allow continuous exchange of influ- participate in important interactions such as cell adhesion, cell
ences, in both directions, between the cytoplasm and material recognition, and the response to protein hormones. As with
in the ECM. lipids, the distribution of membrane polypeptides is different
Membranes range from 7.5 to 10 nm in thickness and in the two surfaces of the cell membranes. Therefore, all mem-
consequently are visible only in the electron microscope. The branes in the cell are asymmetric.
line between adjacent cells sometimes seen faintly with the Studies with labeled membrane proteins of cultured cells
light microscope consists of plasma membrane proteins plus reveal that many such proteins are not bound rigidly in place
extracellular material, which together can reach a dimension and are able to move laterally (Figure 2–4). Such observa-
visible by light microscopy. tions as well as data from biochemical, electron microscopic,
Membrane phospholipids are amphipathic, consisting of and other studies showed that membrane proteins comprise
two nonpolar (hydrophobic or water-repelling) long-chain a moveable mosaic within the fluid lipid bilayer, the well-
fatty acids linked to a charged polar (hydrophilic or water- established fluid mosaic model for membrane structure
attracting) head that bears a phosphate group (Figure 2–1a). (Figure 2–2a). Unlike the lipids, however, lateral diffusion of
Phospholipids are most stable when organized into a double many membrane proteins is often restricted by their cyto-
layer (bilayer) with the hydrophobic fatty acid chains located skeletal attachments. Moreover, in most epithelial cells tight
in a middle region away from water and the hydrophilic polar junctions between the cells (see Chapter 4) also restrict lat-
head groups contacting the water (Figure 2–1b). Molecules eral diffusion of unattached transmembrane proteins and
of cholesterol, a sterol lipid, insert at varying densities among outer layer lipids, producing different domains within the
the closely-packed phospholipid fatty acids, restricting their cell membranes.
movements and modulating the fluidity of all membrane Membrane proteins that are components of large enzyme
components. The phospholipids in each half of the bilayer are complexes are also usually less mobile, especially those
different. For example, in the well-studied membranes of red involved in the transduction of signals from outside the cell.
blood cells, phosphatidylcholine and sphingomyelin are more Such protein complexes are located in specialized membrane

02_Mescher_ch02_p017-052.indd 18 25/04/18 6:47 pm

Another random document with
no related content on Scribd:
 This sub-order has been established for the reception of the
curious genus Opilioacarus.
Fam. Opilioacaridae.—Mites with segmented abdomen, leg-like
palps, chelate chelicerae, and two pairs of eyes. There are four dorsal
abdominal stigmata. Four species of the sole genus Opilioacarus
have been recorded, O. segmentatus from Algeria, O. italicus from
Italy, O. arabicus from Arabia, and O. platensis[373] from South
America.
 APPENDICES TO ARACHNIDA
I. and II.

TARDIGRADA AND PENTASTOMIDA

BY

ARTHUR E. SHIPLEY, M.A., F.R.S.

Fellow and Tutor of Christ’s College, Cambridge, and Reader in

Zoology in the University
 CHAPTER XIX
TARDIGRADA

OCCURRENCE—ECDYSIS—STRUCTURE—DEVELOPMENT
—AFFINITIES—BIOLOGY—DESICCATION—PARASITES—
SYSTEMATIC

The animals dealt with in this chapter lead obscure lives, remote
from the world, and few but the specialist have any first-hand
acquaintance with them. Structurally they are thought to show
affinities with the Arachnida, but their connexion with this Phylum is
at best a remote one.
Tardigrades are amongst the most minute multicellular animals
which exist, and their small size—averaging from ⅓ to 1 mm. in
length—and retiring habits render them very inconspicuous, so that
as a rule they are overlooked; yet Max Schultze[374] asserts that
without any doubt they are the most widely distributed of all
segmented animals. They are found amongst moss, etc., growing in
gutters, on roofs, trees or in ditches, and in such numbers that
Schultze states that almost any piece of moss the size of a pea will, if
closely examined, yield some members of this group, but they are
very difficult to see. The genus Macrobiotus especially affects the
roots of moss growing on stones and old walls. M. macronyx lives
entirely in fresh water, and Lydella dujardini and Echiniscoides
sigismundi are marine; all other species are practically terrestrial,
though inhabiting very damp places.
In searching amongst the heather of the Scotch moors for the ova
and embryos of the Nematodes which infest the alimentary canal of
the grouse, I have recently adopted a method not, as far as I am
aware, in use before, and one which in every case has yielded a good
supply of Tardigrades otherwise so difficult to find. The method is to
soak the heather in water for some hours and then thoroughly shake
it, or to shake it gently in a rocking machine for some hours. The
sediment is allowed to settle, and is then removed with a pipette and
placed in a centrifugaliser. A few turns of the handle are sufficient to
concentrate at the bottom of the test-tubes a perfectly amazing
amount of cryptozoic animal life, and amongst other forms I have
never failed to find Tardigrades.
Many Tardigrades are very
transparent; their cells are large,
and arranged in a beautifully
symmetrical manner; and since
those of them that live in moss,
and at times undergo desiccation,
are readily thrown into a perfectly
motionless state, during which
they may be examined at leisure,
it is not surprising that these little
creatures have been a favourite
object for histological research.
One way to produce the above-
mentioned stillness is partly to
asphyxiate the animals by placing
them in water which has been
boiled, and covering the surface
of the water with a film of oil.
The whole body is enclosed in a
thin transparent cuticle, which
must be pierced by a needle if it
be desired to stain the tissues of
the interior. As a rule the cuticle
is of the same thickness all over
the body, but in the genus
Fig. 249.—Dorsal view of Echiniscus
Echiniscus the cuticle of the
testudo, C. Sch., × 200, showing the
four segments 1, 2, 3, 4. (From Doyère.) dorsal surface is arranged in
thickened plates, and these plates
are finely granulated. From time
to time the cuticle is cast, and this is a lengthy process, so that it is
not unusual to find a Tardigrade ensheathed in two cuticles, the
outer of which is being rubbed off. The Macrobioti lay their eggs in
their cast cuticle (Fig. 250). The end of each of the eight legs bears
forked claws of cuticular origin. The legs are not jointed except in the
genus Lydella, where two divisions are apparent.
 Within the cuticle is the
epidermis, a single layer of cells
arranged in regular longitudinal
and transverse rows along the
upper and under surface, where
the cells are as uniformly
arranged and as rectangular as
bricks. The cells on the sides of
the body are polygonal, and not in
such definite rows. The nuclei
show the same diagrammatic
symmetry as the cells which
contain them, and lie in the same
relative position in neighbouring
cells. In a few places, such as the
end of each limb and around the
mouth and arms, the cells of the
epidermis are heaped up and
form a clump or ridge. In some
genera a deposit of pigment in the
epidermis, which increases as the
animal grows old, obscures the
internal structures. It is generally
brown, black, or red in colour.
The cuticle and epidermis
enclose a space in which the
various internal organs lie. This
space is traversed by numerous
symmetrically disposed muscle- Fig. 250.—Cast-off cuticle of
fibres, and contains a clear fluid— Macrobiotus tetradactylus, Gr., ×
the blood—which everywhere about 150, containing four eggs in
which the boring apparatus of the
bathes these organs. This fluid embryo can be distinguished. (From R.
evaporates when desiccation Greeff.)
takes place, and is soon replaced
after rain; it forms no coagulum
when reagents are added to it, and it probably differs but little from
water. Floating in it are numerous corpuscles, whose number
increases with age. In well-fed Tardigrades the corpuscles are packed
with food-reserves, often of the same colour—green or brown—as the
 contents of the stomach, which
soon disappear when the little
creatures are starved.
The alimentary canal begins
with an oral cavity, which is in
many species surrounded by
chitinous rings. The number of
Fig. 251.—Echiniscus spinulosus, C.
these rings and their general
Sch., × about 200, seen from the side. arrangement are of systematic
(From Doyère.) importance. The oral cavity opens
behind into a fine tube lined with
chitin, very characteristic of the
Tardigrada, which has been termed the mouth-tube. By its side,
converging anteriorly, lie the two chitinous teeth, which may open
ventrally into the mouth-tube, as in Macrobiotus hufelandi and
Doyeria simplex, or may open directly into the oral cavity, as in
Echiniscus, Milnesium, and some species of Macrobiotus. In some of
the last named the tips of the teeth are hardened by a calcareous
deposit. The hinder end of each stylet or tooth is supported by a
second chitinous tooth-bearer,[375] and the movement of each is
controlled by three muscles, one of which, running forwards to the
mouth, helps to protrude the tooth, whilst the other two running
upwards and downwards to the sheath of the pharynx, direct in what
plane the tooth shall be moved.
The mouth-tube passes suddenly into the muscular sucking
pharynx, which is pierced by a continuation of its chitinous tube.
Roughly speaking, the pharynx is spherical; the great thickness of its
walls is due to radially arranged muscles which run from the
chitinous tube to a surrounding membrane. When the muscles
contract, the lumen of the tube is enlarged, and food, for the most
part liquid, is sucked in. Two large glands, composed of cells with
conspicuous nuclei, but with ill-defined cell outlines, pour their
contents into the mouth in close proximity to the exit of the teeth.
The secretion of the glands—often termed salivary glands—is said in
many cases to be poisonous.
The pharynx may be followed by a distinct oesophagus, or it may
pass almost immediately into the stomach, which consists of a layer
of six-sided cells arranged in very definite rows. In fully-fed
specimens these cells project into
the lumen with a well-rounded
contour. Posteriorly the stomach
contracts and passes into the
narrow rectum, which receives
anteriorly the products of the
excretory canals and the
reproductive organs, and thus
forms a cloaca. Its transversely
placed orifice lies between the last
pair of legs. The food of
Tardigrades is mainly the sap of
mosses and other humble plants,
the cell-walls of which are pierced
by the teeth of the little creatures.
The organs to which an
excretory function has been
attributed are a pair of lateral
caeca, which vary much in size
according as the possessor is well
or ill nourished. They recall the
Malpighian tubules of such Mites
as Tyroglyphus. Nothing
comparable in structure to
nephridia or to coxal glands has
been found. Fig. 252.—Macrobiotus schultzei, Gr.,
The muscles show a beautiful × 150. (Modified from Greeff.) a, The
symmetry. There are ventral, six inner papillae of the mouth; b, the
dorsal, and lateral bundles, and chitin-lined oesophagus; c, calcareous
others that move the limbs and spicule; d, muscle which moves the
spicule; e, muscular pharynx with
teeth, but the reader must be masticating plates; f, salivary glands; g,
referred to the works of Basse, stomach; h, ovary; i, median dorsal
Doyère,[376] and Plate[377] for the accessory gland; k, diverticula of
details of their arrangement. The rectum.
muscle-fibres are smooth.
The nervous system consists of a brain or supra-oesophageal
ganglion, whose structure was first elucidated by Plate, and a ventral
chain of four ganglia. Anteriorly the brain is rounded, and gives off a
 nerve to the skin; posteriorly each
half divides into two lobes, an
inner and an outer. The latter
bears the eye-spot when this is
present. Just below this eye a
slender nerve passes straight to
the first ventral ganglion. The
brain is continued round the oral
cavity as a thick nerve-ring, the
ventral part of which forms the
sub-oesophageal ganglion, united
by two longitudinal commissures
Fig. 253.—Brain of Macrobiotus to the first ventral ganglion. Thus
hufelandi, C. Sch., × about 350. (From the brain has two channels of
Plate.) Seen from the side. ap, Lobe of communication between it and
brain bearing the eye; ce, supra- the ventral nerve-cord on each
oesophageal ganglion; d, tooth; Ga,
first ventral ganglion; ga’, sub-
side, one by means of the slender
oesophageal ganglion; k, thickening of nerve above mentioned, and one
the epidermis round the mouth; oc, through the sub-oesophageal
eye-spot; oe, oesophagus; op, nerve ganglion. The ventral chain is
running from the ocular lobe of the composed of four ganglia
brain to the first ventral ganglion; ph, connected together by widely
pharynx. divaricated commissures. Each
ganglion gives off three pairs of
nerves, two to the ventral musculature, and one to the dorsal. The
terminations of these nerves in the muscles are very clearly seen in
these transparent little creatures, though there is still much dispute
as to their exact nature.
The older writers considered the Tardigrada as hermaphrodites,
but Plate and others have conclusively shown that they are bisexual,
at any rate in the genus Macrobiotus. The males are, however, much
rarer than the females. The reproductive organs of both sexes are
alike. Both ovary and testis are unpaired structures opening into the
intestine, and each is provided with a dorsal accessory gland placed
near its orifice. In the ovary many of the eggs are not destined to be
fertilised, but serve as nourishment for the more successful ova
which survive.
 No special circulatory or respiratory organs exist, and, as in many
other simple organisms, there is no connective tissue.
The segmentation of the egg in
M. macronyx is total and equal,
according to the observations of
von Erlanger.[378] A blastula,
followed by a gastrula, is formed.
The blastopore closes, but later
the anus appears at the same
spot. There are four pairs of
mesodermic diverticula which
give rise to the coelom and the
chief muscles. The reproductive
organs arise as an unpaired
diverticulum of the alimentary
canal, which also gives origin to
the Malpighian tubules. The
development is thus very
primitive and simple, and affords
no evidence of degeneration. Fig. 254.—Male reproductive organs of
With regard to their position in Macrobiotus hufelandi, C. Sch., ×
about 350. (From Plate.) a.ep,
the animal kingdom, writers on Epidermal thickening round anus; cl,
the Tardigrada are by no means cloaca; gl.d, accessory gland; gl.l,
agreed. O. F. Müller placed them Malpighian gland; st, stomach; te,
with the Mites; Schultze and testis; x, mother-cells of spermatozoa.
Ehrenberg near the Crustacea;
Dujardin and Doyère with the
Rotifers near the Annelids; and von Graff with the Myzostomidae
and the Pentastomida. Plate regards them as the lowest of all air-
breathing Arthropods, but he carefully guards himself against the
view that they retain the structure of the original Tracheates from
which later forms have been derived. He looks upon Tardigrades as a
side twig of the great Tracheate branch, but a twig which arises
nearer the base of the branch than any other existing forms. These
animals seem certainly to belong to the Arthropod phylum,
inasmuch as they are segmented, have feet ending in claws,
Malpighian tubules, and an entire absence of cilia. The second and
third of these features indicate a relationship with the Tracheate
groups; on the other hand there is an absence of paired sensory
appendages, and of mouth-parts. Von Erlanger has pointed out that
the Malpighian tubules, arising as they do from the mid-gut, are not
homologous with the Malpighian tubules of most Tracheates, and he
is inclined to place this group at the base or near the base of the
whole Arthropod phylum. They, however, show little resemblance to
any of the more primitive Crustacea. The matter must remain to a
large extent a matter of opinion, but there can be no doubt that the
Tardigrades show more marked affinities to the Arthropods than to
any other group of the animal kingdom.
Biology.—Spallanzani, who published in the year 1776 his
Opuscules de physique animale et végétale, was the first
satisfactorily to describe the phenomena of the desiccation of
Tardigrades, though the subject of the desiccation of Rotifers,
Nematodes, and Infusoria had attracted much notice, since
Leeuwenhoek had first drawn attention to it at the very beginning of
the century. In its natural state and in a damp atmosphere
Tardigrades live and move and have their being like other animals,
but if the surroundings dry up, or if one be isolated on a microscopic
slide and slowly allowed to dry, its movements cease, its body
shrinks, its skin becomes wrinkled, and at length it takes on the
appearance of a much weathered grain of sand in which no parts are
distinguishable. In this state, in which it may remain for years, its
only vital action must be respiration, and this must be reduced to a
minimum. When water is added it slowly revives, the body swells,
fills out, the legs project, and gradually it assumes its former plump
appearance. For a time it remains still, and is then in a very
favourable condition for observation, but soon it begins to move and
resumes its ordinary life which has been so curiously interrupted.
All Tardigrades have not this peculiar power of revivification—
anabiosis, Preyer calls it—it is confined to those species which live
amongst moss, and the process of desiccation must be slow and,
according to Lance,[379] the animal must be protected as much as
possible from direct contact with the air.
According to Plate, the Tardigrada are free from parasitic Metazoa,
which indeed could hardly find room in their minute bodies. They
are, however, freely attacked by Bacteria and other lowly vegetable
organisms, and these seem to flourish in the blood without
apparently producing any deleterious effects on the host. Plate also
records the occurrence of certain enigmatical spherical bodies which
were found in the blood or more usually in the cells of the stomach.
These bodies generally appeared when the Tardigrades were kept in
the same unchanged water for some weeks. Nothing certain is known
as to their nature or origin.
Systematic.—A good deal of work has recently been done by Mr.
James Murray on the Polar Tardigrades and on the Tardigrades of
Scotland, many of which have been collected by the staff of the Lake
Survey.[380] Over forty species have been described from North
Britain.
The following table of Classification is based on that drawn up by
Plate:—
 Table of Genera.
I. The claws of the legs are simple, without a second hook. If there
are several on the same foot they are alike in structure and size.
A. The legs are short and broad, each with at least two claws.

2–4 claws Gen. 1. ECHINISCUS, C. Sch. (Fig. 249).

7–9 claws Sub-gen. 1a. ECHINISCOIDES, Plate.

B. The legs are long and slender; each bears only one small
claw.

Gen. 2. LYDELLA, Doy.

II. The claws of the legs are all or partly two- or three-hooked.
Frequently they are of different lengths.
A. There are no processes or palps around the mouth.
I. The muscular sucking pharynx follows closely on the
mouth-tube.
α. The oral armature consists on each side of a stout
tooth and a transversely placed support.

Gen. 3. MACROBIOTUS, C. Sch.

(Fig. 252).

β. The oral armature consists on each side of a stylet-

like tooth without support.

Gen. 4. DOYERIA, Plate.

II. The mouth-tube is separated from the muscular

sucking pharynx by a short oesophagus.

Gen. 5. DIPHASCON, Plate (Fig.

255).
 B. Six short processes or palps surround the mouth, and
two others are placed a little farther back.

Gen. 6. MILNESIUM, Doy.

1. Genus ECHINISCUS (= EURYDIUM, Doy.).—The dorsal cuticle

is thick, and divided into a varying number of shields, which bear
thread- or spike-like projections. The anterior end forms a proboscis-
like extension of the body. Two red eye-spots. There are many
species, and the number has increased so rapidly in the last few years
that specialists are talking of splitting up the genus. E. arctomys,
Ehrb.; E. mutabilis, Murray; E. islandicus, Richters; E. gladiator,
Murray; E. wendti, Richters; E. reticulatus, Murray; E. oihonnae,
Richters; E. granulatus, Doy.; E. spitzbergensis, Scourfield;[381] E.
quadrispinosus, Richters; and E. muscicola, Plate, are all British.
More than one-half of these species are also Arctic, and E. arctomys
is in addition Antarctic. In fact, the group is a very cosmopolitan one.
The genus is also widely distributed vertically, specimens being
found in cities on the sea level and on mountains up to a height of
over 11,000 feet.
1a. Sub-genus ECHINISCOIDES differs from the preceding in the
number of the claws, the want of definition in the dorsal plates, and
in being marine. The single species E. sigismundi, M. Sch., is found
amongst algae in the North Sea (Ostend and Heligoland).
2. Genus LYDELLA.[382]—The long, thin legs of this genus have two
segments, and in other respects approach the Arthropod limb.
Marine. Plate suggests the name L. dujardini for the single species
known.
3. Genus MACROBIOTUS has a pigmented epidermis, but eye-
spots may be present or absent. The eggs are laid one at a time, or
many leave the body at once. They are either quite free or enclosed in
a cast-off cuticle. The genus is divided into many species and shows
signs of disruption. They mostly live amongst moss; but M.
macronyx, Doy., is said to live in fresh water. The following species
are recorded from North Britain: M. oberhäuseri, Doy.; M.
hufelandi, Schultze; M. zetlandicus, Murray; M. intermedius, Plate;
M. angusti, Murray; M. annulatus, Murray; M. tuberculatus, Plate;
M. sattleri, Richters; M. papillifer, Murray; M. coronifer, Richters;
 M. crenulatus, Richters; M.
harmsworthi, Murray; M.
orcadensis, Murray; M.
islandicus, Richters; M. dispar,
Murray; M. ambiguus, Murray;
M. pullari, Murray; M. hastatus,
Murray; M. dubius, Murray; M.
echinogenitus, Richters; M.
ornatus, Richters; M. macronyx?,
Doy.
4. Genus DOYERIA.—The teeth
of this genus have no support,
and the large salivary glands of
the foregoing genus are absent; in
other respects Doyeria, with the
single species Doyeria simplex,
Plate, resembles Macrobiotus,
and is usually to be found in
consort with M. hufelandi, C. Sch.
5. Genus DIPHASCON
resembles M. oberhäuseri, Doy.,
but an oesophagus separates the
mouth-tube from the sucking
pharynx, and the oral armature is
weak. The following species are
British, the first named being
very cosmopolitan, being found at
both Poles, in Chili, Europe, and
Asia: D. chilenense, Plate; D.
Fig. 255.—Diphascon chilenense, Plate, scoticum, Murray; D. bullatum,
× about 100. (From Plate.) ce, Brain; k, Murray; D. angustatum, Murray;
thickening of the epidermis above the
mouth; o, egg; oe, oesophagus; p,?
D. oculatum, Murray; D.
salivary glands; ph, pharynx; sa, blood alpinum, Murray; D.
corpuscles; st, stomach. spitzbergense, Murray.
6. Genus MILNESIUM has a
soft oral armature, and the teeth
open straight into the mouth. A lens can usually be distinguished in
the eyes. Two species have been described, M. tardigradum, Doy.,
British, and M. alpigenum, Ehrb. Bruce and Richters consider that
these two species are identical.
 CHAPTER XX
[383]
PENTASTOMIDA

OCCURRENCE—ECONOMIC IMPORTANCE—STRUCTURE
—DEVELOPMENT AND LIFE-HISTORY—SYSTEMATIC

Pentastomids are unpleasant-looking, fluke-like or worm-like

animals, which pass their adult lives in the nasal cavities, frontal
sinuses, and lungs of flesh-eating animals, such as the Carnivora,
Crocodiles, and Snakes; more rarely in Lizards, Birds, or Fishes.
From these retreats their eggs or larvae are sneezed out or coughed
up, or in some other way expelled from the body of their primary
host, and then if they are eaten, as they may well be if they fall on
grass, by some vegetable-feeding or omnivorous animal, they
undergo a further development. If uneaten the eggs die. When once
in the stomach of the second host, the egg-shell is dissolved and a
larva emerges (Fig. 260, p. 494), which bores through the stomach-
wall and comes to rest in a cyst in some of the neighbouring viscera.
Here, with occasional wanderings which may prove fatal to the host,
it matures, and should the second host be eaten by one of the first,
the encysted form escapes, makes its way to the nasal chambers or
lungs, and attaching itself by means of its two pairs of hooks, comes
to rest on some surface capable of affording nutriment. Having once
taken up its position the female seldom moves, but the males, which
are smaller than the females, are more active. They move about in
search of a mate. Further, should the host die, both sexes, after the
manner of parasites, attempt to leave the body. Like most animals
who live entirely in the dark they develop no pigment, and have a
whitish, blanched appearance.
The only species of Pentastomid which has any economic
importance is Linguatula taenioides of Lamarck, which is found in
the nose of the dog, and much more rarely in the same position in
the horse, mule, goat, sheep, and man. It is a comparatively rare
parasite, but occurred in about 10 per cent of the 630 dogs in which
it was sought at the laboratory of Alfort, near Paris, and in 5 out of
60 dogs examined at Toulouse. The symptoms caused by the
presence of these parasites are not usually very severe, though cases
have been recorded where they have caused asphyxia. The larval
stages occur in the rabbit, sheep, ox, deer, guinea-pig, hare, rat,
horse, camel, and man, and by their wandering through the tissues
may set up peritonitis and other troubles.
As in the Cestoda, which they so closely resemble in their life-
history, the nomenclature of the Pentastomids has been complicated
by their double life. For long the larval form of L. taenioides was
known by different names in different hosts, e.g. Pentastoma
denticulatum, Rud., when found in the goat, P. serratum, Fröhlich,
when found in the hare, P. emarginatum when found in the guinea-
pig, and so on. In the systematic section of this article some of the
species mentioned are known in the adult state, some in the larval,
and in only a few has the life-history been fully worked out.
Structure.[384]—The body of a Pentastomid is usually white,
though in the living condition it may be tinged red by the colour of
the blood upon which it lives. The anterior end, which bears the
mouth and the hooks (Fig. 256), has no rings; this has been termed
the cephalothorax. The rest of the body, sometimes called the
abdomen, is ringed, and each annulus is divided into an anterior half
dotted with the pores of certain epidermal glands and a hinder part
of the ring in which these are absent.
On the ventral surface of the cephalothorax, in the middle line, lies
the mouth, elevated on an oral papilla, and on each side of the mouth
are a pair of hooks whose bases are sunk in pits. The hooks can be
protruded from the pits, and serve as organs of attachment. Their
shape has some systematic value.
There are a pair of peculiar papillae which bear the openings of the
“hook-glands,” lying just in front of the pairs of hooks, and other
smaller papillae are arranged in pairs on the cephalothorax and
anterior annuli. The entire body is covered by a cuticle which is
tucked in at the several orifices. This is secreted by a continuous
layer of ectoderm cells. Some of these subcuticular cells are
aggregated together to form very definite glands opening through the
cuticle by pores which have somewhat unfortunately received the
name of stigmata. Spencer attributes to these glands a general
excretory function. There is, however, a very special pair of glands,
the hook-glands, which extend almost from one end to the other of
the body; anteriorly these two lateral glands unite and form the
head-gland (Fig. 257). From this
on each side three ducts pass, one
of which opens to the surface on
the primary papilla; the other two
ducts open at the base of the two
hooks which lie on each side of
the mouth. Leuckart has
suggested that these important
glands secrete some fluid like the
irritating saliva of a Mosquito
which induces an increased flow
of blood to the place where it is of
use to the parasite. Spencer,
however, regards the secretion as
having, like the secretion of the
so-called salivary cells of the
Leech, a retarding action on the
coagulation of the blood of the
host.
The muscles of Pentastomids
are striated. There is a circular
layer within the subcuticular
cells, and within this a
longitudinal layer and an oblique
layer which runs across the body-
cavity from the dorso-lateral
surface to the mid-ventral line, a Fig. 256.—Porocephalus annulatus,
primitive arrangement which Baird. A, Ventral view of head, × 6; B,
recalls the similar division of the ventral view of animal, × 2.
body-cavity into three chambers
in Peripatus and in many Chaetopods. Besides these there are
certain muscles which move the hooks and other structures.
The mouth opens into a pharynx which runs upwards and then
backwards to open into the oesophagus (Fig. 257). Certain muscles
attached to these parts enlarge their cavities, and thus give rise to a
sucking action by whose force the blood of the host is taken into the
alimentary canal. The oesophagus opens by a funnel-shaped valve
into the capacious stomach or mid-gut, which stretches through the
body to end in a short rectum or hind-gut. The anus is terminal.

Fig. 257.—Diagrammatic representation of the alimentary,

secretory, nervous, and reproductive systems of a male
Porocephalus teretiusculus, seen from the side. The nerves are
represented by solid black lines. (From W. Baldwin Spencer.)

1, Head-gland; 2, testis; 3, hook-gland; 4, hind-gut; 5, mid-gut; 6,

ejaculatory
duct; 7, vesicula seminalis; 8, vas deferens; 9, dilator-rod sac; 10,
cirrus-bulb;
11, cirrus-sac; 12, fore-gut; 13, oral papillae.

There appears to be no trace of circulatory or respiratory organs,

whilst the function usually exercised by the nephridia or Malpighian
tubules or by coxal glands, of removing waste nitrogenous matter,
seems, according to Spencer, to be transferred to the skin-glands.
The nervous system is aggregated into a large ventral ganglion
which lies behind the oesophagus. It gives off a narrow band devoid
of ganglion-cells, which encircles that tube. It also gives off eight
nerves supplying various parts, and is continued backward as a ninth
pair of prolongations which, running along the ventral surface, reach
almost to the end of the body (Fig. 257). The only sense-organs
known are certain paired papillae on the head, which is the portion
that most closely comes in contact with the tissues of the host.
Pentastomids are bisexual. The males are as a rule much less
numerous and considerably smaller than the females, although the
number of annuli may be greater.
The ovary consists of a single tube closed behind. This is supported
by a median mesentery. Anteriorly the ovary passes into a right and
left oviduct, which, traversing the large hook-gland, encircle the
alimentary canal and the two posterior nerves (Fig. 258). They then
unite, and at their point of union they receive the ducts of the two
spermathecae, usually found packed with spermatozoa. Having
received the orifices of the spermatheca, the united oviducts are
continued backward as the uterus, a highly-coiled tube in which the
fertilised eggs are stored. These are very numerous; Leuckart
estimated that a single female may contain half a million eggs. The
uterus opens to the exterior in the mid-ventral line a short distance—
in P. teretiusculus on the last ring but seven—in front of the terminal
anus. In L. taenioides the eggs begin to be laid in the mucus of the
nose some six months after the parasite has taken up its position.

Fig. 258.—Diagrammatic representation of the alimentary,

secretory, nervous, and reproductive systems of a female
Porocephalus teretiusculus, seen from the side. The nerves are
represented by solid black lines. (From W. Baldwin Spencer.)

1, Head-gland; 2, oviduct; 3, hook-gland; 4, mid-gut; 5, ovary; 6,

hind-gut; 7,
vagina; 8, uterus; 9, accessory gland; 10, spermatheca.

The testis is a single tube occupying in the male a position similar

to that of the ovary in the female. Anteriorly it opens into two
vesiculae seminales, which, like the oviducts, pierce the hook-glands
and encircle the alimentary canal (Fig. 257). Each vesicula passes
into a vas deferens with a cuticular lining. Each vas deferens also
receives the orifice of a muscular caecal ejaculatory duct, which,
crowded with mature spermatozoa, stretches back through the body.
Anteriorly the vas deferens passes into a cirrus-bulb, which is joined
by a cirrus-sac on one side and a dilator-rod sac on the other,
structures containing organs that assist in introducing the
spermatozoa into the female. The two tubes then unite, and having