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THE ART OF EXAMINING AND INTERPRETING HISTOLOGIC PREPARATIONS: A LABORATORY MANUAL AND STUDY GUIDE FOR HISTOLOGY Second Edition

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THE ART OF EXAMINING AND INTERPRETING HISTOLOGIC
PREPARATIONS: A LABORATORY MANUAL AND STUDY GUIDE FOR HISTOLOGY
Second Edition
William J. Krause
Department of Pathology and Anatomical Sciences
School of Medicine
Columbia, Missouri
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TABLE OF CONTENTS
Introduction 3
Chapter 1 Getting started 4
Chapter 2 Epithelium 8
Chapter 3 General connective tissue 15
Chapter 4 Specialized connective tissue 19
Chapter 5 Muscle tissue 23
Chapter 6 Nerve tissue 25
Chapter 7 Peripheral blood & bone marrow 30
Chapter 8 Cardiovascular system 37
Chapter 9 Lymphatic organs 42
Chapter 10 Integument 47
Chapter 11 Digestive system 50
Chapter 12 Respiratory system 61
Chapter 13 Urinary system 66
Chapter 14 Male reproductive system 70
Chapter 15 Female reproductive system 75
Chapter 16 Classic endocrine glands 81
Chapter 17 Organs of special sense 85
Appendix Tables 91
Index 98
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INTRODUCTION
The examination and interpretation of tissue sections
seen under the light microscope in a laboratory setting
is an example of student-directed, independent
problem solving. The proper reading of a histologic
section is an acquired art that can only be developed
through practice, close observation and repetition.
This laboratory manual was designed as a guide for
students to aid them in this endeavor. The laboratory
study guide/manual was designed to be used as a
supplement to any current textbook and/or atlas of
Histology. Learning objectives provide the overall
goals for each chapter. The narrative of the study
guide explains how to systematically breakdown,
examine and interpret each tissue and/or organ
encountered, without regard to a given histologic slide
from a specific slide collection. Thus, this systematic
method can be used to examine and interpret
histologic preparations from any collection or of any
species.
The student is encouraged to sketch, label and
create a personalized atlas while using this
laboratory manual as a guide. The vocabulary that
should be developed and used during the laboratory
can be found quickly by going to the bold face type in
the appropriate segment of the text. Each chapter
contains one or more tables in which key structures
used in the identification of a tissue/organ are
presented, offering the briefest possible summary of
important histologic features. As a final short review,
an appendix provides summary tables that compare
and contrasts the basic differences of several
structures that are somewhat similar in general
architecture. William J. Krause
Department of Pathology and
Anatomical Sciences, School of Medicine,
University of Missouri-Columbia, Columbia, MO
August 2004
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CHAPTER 1. GETTING STARTED
Have an appreciation of how a histologic
preparation is made
The human body consists of two basic components:
cells and products of cells (extracellular materials).
The discipline of histology is concerned primarily with
the microscopic examination of these two
components and how they are organized into the
various tissues and organs of the body.
Obviously, if the liver, or a similar organ, were
to be examined, it would be impractical to place the
entire organ under a routine light microscope for
study. It is not only much too large, but also opaque,
therefore an examination of its microcomponents
would be impossible. For this reason, and several
others, a small portion of a specific tissue or organ
must be excised from a given organ and processed
for microscopic analysis. The excised tissue is placed,
as soon as possible after removal, into a reagent
known as a fixative. Fixatives act to preserve the cells
and extracellular substances of tissues/organs and
prevent autolytic (degenerative) changes. Although
there are numerous fixatives developed for a variety of
purposes, 10% buffered formalin is one of the most
commonly used, routine fixatives in biology, medicine
(surgical and general pathology) and biomedical
research. The collected tissues, once fixed, are then
dehydrated in graded solutions of alcohol or other
dehydrating agents. Following removal of the majority
of water from the collected specimens during
dehydration, the tissues are cleared. Clearing is the
process of removing the dehydrating agent and
replacing it with a fluid that is miscible both with the
dehydrating agent used and with the type of
embedding medium chosen to make the tissue sample
firm throughout. As with dehydrating agents, there are
a large number of clearing reagents, the selection of
which is dependent largely on the embedding medium
chosen. Xylene and toluene are in common use for
paraffin embedding; propylene oxide for embedding in
several of the plastic embedding media. The tissue
sample is next infiltrated with and embedded in the
chosen embedding medium so that a firm
homogeneous mass of material containing the tissue
sample is obtained. Paraffin is the most commonly
used embedding medium for routine preparations.
The formed paraffin block, together with the
contained tissue, is then sectioned (cut) into very thin
slices called sections that normally range between 4
and 7 microns (µm) in thickness. The instrument used
in cutting histologic sections is called a microtome.
The cut sections are then transferred (mounted) onto
the surface of clean glass microscope slides.
In order to prepare the mounted sections for
staining, the paraffin embedding medium must be
removed. This is accomplished by passing the slides
together with their mounted sections through xylene
or toluene to remove the paraffin and then through
descending strengths of alcohol solutions to water, as
most dyes used are in aqueous solutions. Staining is a
process of increasing the visibility of cells by the
application of dyes or by the reaction of chemical
reagents with the tissue components to form visible
substances. A large number of stains are available but
generally only two stains are used together to provide
contrasting color: one to stain the cytoplasm of cells,
the other to stain the nuclei. The most common and
universally used combination is the hematoxylin and
eosin (H&E) stain. When this stain is applied to a
section of tissue the nuclei of component cells appear
blue; the cytoplasm and most extracellular
materials a light pink-orange. Staining is necessary
because the vast majority of cells and their
extracellular materials are transparent and lack color.
Only naturally occurring pigment granules such as
melanin and lipofuscin would be visible on
examination. The color of the dyes used during
staining markedly increases the contrast of cells, their
sub-components, and the associated extracellular
materials. Without staining, the examination of cells
and tissues with a routine light microscope would be
extremely difficult. Following staining in aqueous dyes,
the slides, together with their mounted, stained
sections, are passed back through ascending
concentrations of alcohol for dehydration, cleared
with some solvent (usually xylene or toluene), and
then a permanent mounting medium is put on the
tissue section. A thin glass cover slip is then placed on
the covering mounting medium and underlying tissue
section and allowed to dry. As the histological
preparation dries, the solvent evaporates from the
mounting medium, which hardens, permanently
cementing and sealing the tissue preparation between
the glass slide and overlying cover slip. The mounting
medium (balsam, damar, Permount) when dried has
nearly the same refractive index as glass. After drying,
the histologic section is well protected and if stored
properly will usually remain unchanged for several
years.
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Know what you are looking for
Before examining a histologic preparation of any given
tissue or organ, know something of the structure
before studying it under the microscope. Such
familiarity is usually acquired by carefully considering
the details of the tissue/organ in question and
formulating a mental image of how the structure
should appear. The microscope should then be used to
confirm or refute the preconceived image
conceptualized. Examination of tissues and organs
without prior thought and consideration of the subject
usually proves frustrating and is often a waste of
considerable time. Therefore, before attempting to
examine any specimen for the first time with the
microscope, know as much about the structure of the
subject matter as possible. This information can only
be acquired by attending lectures and/or reading
textbooks and studying atlases.
It is highly recommended that a small
labeled sketch be made of each section examined
under the microscope using colored pencils, noting
relationships and the position of specific structures
and cells. This simple exercise aids in focusing
concentration on the structure(s) being examined and
avoids casual observation. The construction of such a
labeled, personalized atlas aids in cementing the
observations made in one's memory and is important
in beginning to develop a mental three-dimensional
image (understanding) of the tissues/organs examined
from the two-dimensional image presented by the
histologic preparation. The labeled sketch also serves
as a highly personalized map of a specific slide for re-
examination later in the exercise and is excellent for
review purposes.
Making sketches
A variety of sketches should be used dependent on the
structural detail needed to clearly understand a given
topic. Use of several different types of sketches is
suggested, the one chose dependent on the needs of
the exercise.
For example: in the first exercise
recommended (the identification of large cells), a
simple line sketch of the entire tissue section can be
used with a label showing precisely which region was
used to look for and examine cells. In the case of the
spinal cord, a simple outline of the tissue is all that is
needed, in which nerve cells (neurons) should be
identified. A supplemental sketch depicting and
labeling the salient points of a neuron (cell shape,
cytoplasm, position and shape of the nucleus,
nucleolus and nuclear envelope) should also be used.
The other example suggested for this very important
initial exercise is the identification of another of the
extraordinarily large cells (ova) in the ovary.
Take care to note their position in this organ and then
examine the surrounding tissue for additional cells that
will show a variety of different sizes and shapes. In
this case, a more detailed sketch should be used to
illustrate the latter points as well as focusing on the
most important aspect of the exercise: being able
to distinguish clearly between the nucleus and the
cytoplasm of a given cell and to estimate nuclear and
cell boundaries.
Later, sketches can be used to illustrate how
cells are organized into units and how understanding
such organization has led to various classification
schemes, as with the classification of epithelial tissues.
In this case, greater detail should be employed to
illustrate cell shape, size and organization. Further
details, such as modification of the cell membrane
(plasmalemma) or attachment points, should also be
noted.
When the histologic makeup of entire organs
is considered, sketches with less cellular detail are
often useful as guides as to where a particular type of
tissue is located in a given organ. This is particularly
true of tubular structures, the walls of which are
formed by several different layers or strata. Additional
accompanying detailed sketches may be needed if an
important characteristic group of cells or other
structures are present within the organ.
Reliable mechanics on how to examine histologic
preparations (slides)
When presented with a histologic preparation (slide),
the very first exercise that needs to be done is to
examine it closely with the naked eye. A
considerable amount of important information can be
ascertained about the preparation using this simple
exercise before placing the slide under the
microscope. Indeed, this exercise is of such
importance it should become part of the "standard
operating procedure" for each slide examined. Initially
pick up the histologic slide between the thumb and
index finger and examine it by holding it up to the
light or against a white background. What to look for:
First, look at the overall nature of the preparation.
Does the preparation have a doughnut configuration?
If so, this immediately suggests to the viewer that the
specimen being examined is tubular in nature and is
being viewed in a transverse profile (hollow organs,
such as blood vessels, regions of the digestive tube,
tubular components of respiratory, urinary and
reproductive systems, are possible organs and this list
can be restricted even further if dealing only with
human tissues by the size of the preparation.
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Identification of the luminal surface (the lining of the
internal space) as well as the external surface will be of
importance when this preparation is examined further.
Does the preparation have a uniform consistency and
appear as a solid mass of tissue cut in the shape of a
square, rectangle or wedge? If so, such a preparation
usually indicates that the sample of tissue was taken
from a large compact (solid) organ such as the liver,
spleen, kidney or pancreas to list just a few. Once a
determination has been made with regard to the shape
and consistency of the tissue mounted on the slide,
then examine it more carefully with regard to its
staining characteristics. Of particular importance is to
note if one surface (external edge or luminal surface of
the doughnut-shaped configuration) stains more
basophilic (light blue) than any other region in the
sample of tissue being examined. Such basophilic
staining usually indicates a high concentration of
nuclei per unit area (nuclei stain blue with hematoxylin
dye). In this way, one of the basic tissues, epithelium,
can usually be located even before the histologic slide
is ever viewed under the microscope. Are additional
small tubular or round structures present within the
tissue sample? These may indicate small blood vessels,
ducts or glandular structures.
Always begin the initial examination of a
histologic preparation with the low-power
(scanning) objective and carefully view the entire
section. This opportunity should be used to confirm
or deny the observations and speculations made by
direct observation with the naked eye. Add more
details to the mental image being developed with
regard to the preparation under examination. Note the
presence or absence of more than one tissue type,
patches of deeper staining, other structures present,
their locations and relationships to one another and to
surfaces. Only after a thorough examination with the
low-power objective should the intermediate- and
high-power objectives be used. Of these, the medium-
power objective is the more useful for study, although
more detail can be seen with the high-power objective.
The disadvantage of the high-power objective is the
smaller field of view and, because of this, relationships
between tissues are often lost. The oil objective, if
used at all, should be used only in the examination of
peripheral blood and bone marrow preparations.
Since the tissues and organs of the body
consist only of two elements, cells and cell products
(extracellular materials) both deserve careful and
thorough study. The initial exercise should be to
examine the morphology of a cell. The following
observations should be made during the examination
of various cell types.
1. Shape of the cell.
2. Size of the cell (determine the position of the cell
membrane).
3. Shape, size and position of the nucleus.
4. Identify the nucleolus if present in the nucleus.
It is absolutely essential that the boundary of
the cell and that of the nucleus be clearly defined.
Examine several cell types of various sizes and shapes
to make these observations.
Large cells
Examine a section of ovary for ova. These are located
in the ovarian cortex at the periphery of the ovary.
The ovum represents a very large, round cell. Because
of their large size, these light-staining cells can be
found, by using the low-power objective, near the
periphery of the ovary. After examining an ovum at
low power, study it further using increased
magnification. Note again the large size of the ovum,
the abundant light-staining cytoplasm, and the central
round nucleus separated from the cytoplasm by a well-
defined nuclear membrane (envelope). Identify the
nucleolus. It is usually round in profile and stains
intensely. The nuclei of several ova may have to be
examined, as these cells are so large that the plane of
section may not pass through the nuclear region
containing the nucleolus in all ova. Examine the
remainder of the ovary and note the differences in the
size and shape of the different cell types. Note that the
nuclear shapes most often assume the shape of the
cells being examined and that the cell membrane of
most cells cannot be resolved with the light
microscope. Therefore, when examining a number of
tissues and organs the nuclei of component cells are
often relied on in determining the orientation and
shape of the cellular component and the cytoplasmic
boundaries of a give cell type are estimated. The shape
of the ovum is generally spherical, that of surrounding
cells cube-shaped, whereas more distant cells in the
ovary are spindle in shape. Examine the nuclear profile
of each group of cells, noting how the shape of the
nucleus reflects the shape of the cell. In addition, note
the dark-staining, clumped nature of the chromatin is
some nuclei. This material is referred to as
heterochromatin and often lies adjacent to the
nuclear envelope. The lighter-staining nuclear material
is referred to as euchromatin.
The next histologic preparation that should be
examined at this time is a transverse section through
the spinal cord. Visual examination of the preparation
will reveal an H-shaped area (gray matter) near the
center of the preparation that surrounds a small
central canal.
7
Examine the gray matter with the low-power objective
and locate numerous, large neurons (nerve cells) found
in the ventral horn. These too are exceptionally large
cells with several irregularly shaped, elongated
processes. Make a clear distinction between the
cytoplasm, nucleus and nucleolus.
Make a small labeled sketch of several cells from these
preparations illustrating the cytoplasm, the size and shape of the
nucleus and the position of the nucleolus.
Cytology: structural components of a cell that can
be examined with the light microscope
This laboratory guide briefly presents a method for
examining the cytologic and histologic details of
human morphology utilizing the routine H&E
preparation. This preparation primarily demonstrates
the nucleus and the surrounding cytoplasm of a given
cell. It must be understood, however, that special
staining methods can be used to demonstrate the
majority of organelles, inclusions and components of
the cytoskeleton within a given cell, as well as a variety
of cell products and extracellular materials. These
special staining methods include a variety of dyes,
antibodies and other probes.
Techniques such as immunohistochemistry, in situ
hybridization and autoradiography are powerful
tools in demonstrating structure, secretory products,
and/or messages (mRNA) within cells, as well as cell
receptors not seen with routine preparations. The
study guide focuses primarily on what can be
visualized using the routine H&E preparation unless
otherwise stated.
Know the basics: the basic tissues
The term tissue [French tissu, woven cloth] is defined
as a collection of similar cells and surrounding
extracellular substances that perform related functions.
Four basic tissue types occur and these are woven
together to form the fabric of all organs.
The four basic tissues are: epithelium,
connective tissue, muscle tissue and nervous
tissue. A thorough understanding of each of these
four basic tissues is necessary before beginning an
examination of individual organs or systems.
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CHAPTER 2. EPITHELIUM
Epithelia consist of closely aggregated cells separated
by only minimal amounts of intervening intercellular
substances. Two general categories are recognized: a
lining, barrier or covering type of epithelium
organized into sheets of cells that form barriers and
glandular epithelium modified for secretion. The
sheet (barrier) form of epithelium covers the external
body surface as the epidermis, lines the body cavities
(pleural, pericardial, peritoneal) as well as the lumina of
the cardiovascular, digestive, respiratory and urogenital
systems. Thus, the majority of, if not all, substances
entering or exiting the "substance of the body" must
first cross an epithelial barrier. All epithelia lie on a
basement membrane and are avascular. The basement
membrane appears as a thin interphase between the
epithelium and underlying connective tissue. It
consists of glycoproteins, proteoglycans rich in
heparin sulfate and type IV collagen. Usually the
basement membrane appears only as an interphase in
H&E preparations but can be demonstrated clearly
using special staining techniques. As different types of
epithelium are examined, a mental record should be
established, keeping track of not only where a specific
type of epithelium is found in a given organ but also
its functional ramifications. Later, in the examination
and identification of organs under the microscope, the
identity and location of a specific epithelium is often
a key feature in the identification.
Lining/covering (barrier) form of epithelium
Learning objectives for lining or covering
epithelium:
1. Be able to locate, identify and classify the various
types of epithelia in a given section of histologic
material.
2. Be able to identify the various specializations of the
cell membrane associated with specific epithelia.
Classification
If the covering or lining form of epithelium consists
only of a single layer of cells it is termed simple. If
two or more layers of cells are present, of which the
superficial most-cells do not reach the basement
membrane, the epithelium is classified as stratified.
Once this determination has been made the next step
is to determine the geometric shape of the
superficial-most cells to complete the classification.
Epithelial cells can be divided into three types
according to their geometric shape: squamous (thin,
flat, plate-like cells), cuboidal (height and width of the
cell are approximately equal with the nucleus nearly
touching all surfaces), and columnar (height of cell is
greater than its width). Cells intermediate in height
between cuboidal and columnar also occur and are
referred to as low columnar. Thus, epithelium
consisting of a single layer of cells can be classified as:
simple squamous, simple cuboidal, or simple
columnar. A fourth type of simple epithelium,
pseudostratified columnar, consists of more than
one cell type whose nuclei occur at different levels
falsely suggesting that the epithelium is made up of
two or more layers. All cells of this type of epithelium
reach the basement membrane but not all reach the
luminal surface.
The term endothelium is the specific name
given to the simple squamous epithelium that lines the
cardiovascular and lymph vascular systems. Examine
the luminal (interior) surface of several blood vessels
for this type of epithelium. The name mesothelium is
given to the simple squamous epithelium that lines the
pleural, pericardial and peritoneal cavities. Examine
the external surface of a region of the stomach,
jejunum or ileum for mesothelium. Examine the
luminal surface of each of these organs for simple
columnar epithelium. Note: Prior to examining these
organs under the microscope, each should be
examined with the naked eye. As an epithelium must
lie on one surface or the other, and in the case of the
gut, both, examine each of these surfaces under the
microscope initially with low power for orientation
prior to moving to higher power objectives for a more
detailed examination.
Simple squamous epithelium
Examine the external surface of the stomach or small
intestine. Note that the cells making up this form of
simple squamous epithelium appear extremely
attenuated, with their flattened, dense nuclei separated
by considerable distances. The cytoplasm often
appears only as a thin interphase between nuclei.
Simple columnar epithelium
Examine the luminal surface of the stomach for typical
simple columnar epithelium. Note the basal position
of the oval-shaped nuclei and that the apical cytoplasm
is filled with unstained secretory granules. These can
be visualized more clearly by lowering the intensity of
the light and/or by dropping the condenser of the
microscope. Terminal bars also can be seen in most
preparations between the apices of adjacent cells.
9
They appear as minute, dense-staining short bars and
represent the light microscopic appearance of the
three part junctional complex (zonula occludens,
zonula adherens, macula adherens) seen with the
electron microscope. Examine a textbook illustration
(electron micrograph) of this important junctional
specialization. Examine the luminal surface of a region
of small intestine. Note that it, like the stomach, is
lined by a simple columnar epithelium. Carefully
examine the intestinal lining epithelium and identify
the striated (microvillus) border on the apical
surface. Terminal bars also may be seen between the
apices of cells forming this epithelium, as well as most
other simple columnar or cuboidal epithelia.
Simple cuboidal epithelium
This type of epithelium is best seen in a section of
kidney medulla, which also contains numerous tubules
lined by either a simple columnar or simple squamous
epithelium. Tubules lined by either simple squamous,
simple columnar or simple cuboidal should be
identified and compared. The nuclei of cells in a good
simple cuboidal epithelium should nearly touch apical,
basal and lateral cell membranes. As different tubules
are examined, several examples of "low columnar" will
also be encountered.
Sketch and compare tubules formed by classic examples of the
three types of simple epithelia observed.
Stratified squamous epithelium
Stratified squamous is the most common of the
stratified epithelia. A good example of stratified
squamous is the epidermis of skin. Once again,
examine the slide visually, noting the surface on which
the epithelium rests, then examine it under low power.
Note the presence of additional components of the
skin but do not examine them at this time. Select an
area of epidermis and examine it carefully under
increased magnification, beginning at the basal surface.
It should be quite obvious that this form of epithelium
is multilayered and consists of cells that vary in their
geometric shape. Cells resting on the basement
membrane are columnar in shape whereas those above
assume a more spindle-shaped configuration. In the
outermost (superficial) layers, the cells become flat and
plate-like, ie. squamous. Despite the large number of
cells with different shapes, recall that the classification
scheme remains based on two questions:
1. Are two or more layers of cells present, the
outermost of which is not in contact with the
basement membrane?
2. What is the geometric shape of the superficial-most
cells?
Thus, the classification must be stratified squamous.
In the case of the epidermis the superficial most cells
also undergo a transformation, known as
keratinization. As a result of this process, cells loose
their nuclei and the cytoplasm becomes filled with a
proteinaceous material called keratin. These
transformed dead cells lie immediately above the intact
layer of squamous cells. When this type of surface
feature is present, it is usually incorporated into the
terminology of the classification scheme of the
epithelium. Thus, in the case of the epidermis, the
complete classification of the epithelium would be
keratinized stratified squamous epithelium.
Repeat this exercise examining the epithelium lining
the lumen of the esophagus. This epithelium lacks the
layer of keratin on its luminal surface. Therefore, it is
classified as a non-keratinized (wet) stratified
squamous epithelium. The term wet is often used
when this type of epithelium makes up a portion of a
mucous membrane or lines a moist environment. Note
the presence of intact nuclei in cells comprising the
superficial most layer of this form of stratified
squamous epithelium.
Compare the overall thickness of this epithelium with that of the
epidermis and make a labeled sketch of each.
Stratified cuboidal
This type of epithelium is limited in distribution to
regions of ducts from larger glands where there is a
transition from a simple epithelium into a stratified
epithelium. Stratified cuboidal epithelium also lines the
ducts of sweat glands. Sweat glands are coiled tubes
comprised of epithelial cells that extend from the base
of the epidermis into the tissue of the underlying
dermis and hypodermis. Because of their coiled nature,
a profile of an entire sweat gland is rarely if ever
encountered in sectioned material. Using the low-
power objective, scan the tissue beneath the epidermis
in a section of skin looking for groups of small circular
and oval profiles. These cellular profiles are often
encountered surprisingly deep within the underlying
tissue. Two profiles will be encountered: one light
staining, the other subtlety darker staining. The more
darkly stained portion of the tubule is the duct region
of the sweat gland. Confirm that the duct region
consists of a stratified cuboidal epithelium, two cells
thick, which surrounds a minute central lumen.
Sketch the duct region of a sweat gland to illustrate a stratified
cuboidal epithelium.
10
Stratified columnar
Like stratified cuboidal, stratified columnar is
restricted in its distribution confined primarily to the
cavernous urethra, fornix of the conjunctiva and large
excretory ducts of major glands. This epithelium is
most conveniently observed in the large ducts of the
parotid or submandibular gland. Examine one of these
glands for the ducts only. They can be identified
initially with the low-power objective by scanning the
gland and looking for tubules with a very large luminal
diameter. Once located, examine the wall of the duct
and determine the nature of the lining epithelium. Is it
stratified? Is the epithelium stratified cuboidal or
stratified columnar? Both types will be encountered.
Search the preparation until an example of each is
found.
Make sketches of two large ducts: one lined by stratified
cuboidal, the other lined by stratified columnar.
Pseudostratified columnar
This type of epithelium is a simple form of epithelium
as all component cells are in contact with the
underlying basement membrane, thereby satisfying the
definition of a simple epithelium. The cells vary
considerably in height and not all reach the luminal
surface. As a result, the respective nuclei are found at
different levels within this epithelium and form what
appears to be two or three layers of cells, falsely
suggesting stratification, hence its name.
Pseudostratified columnar epithelium is somewhat
restricted in its distribution, being confined primarily,
but not exclusively, to the conducting portion of the
respiratory system and the excurrent ducts of the male
reproductive system.
Examine the trachea and epididymis for
examples of this type of epithelium. Examine the
luminal surface of the trachea and note the height of
the epithelium and the stratified appearance due to the
position of component nuclei. With the high-power
objective examine the epithelium in detail beginning at
the base noting the different sizes and shapes of cells
forming this epithelium. Scattered within this epithelial
layer are unicellular exocrine glands known as goblet
cells. Goblet cells are sandwiched among the other
epithelial cells and usually have a drinking goblet
(wineglass) shape. The base of this cell is usually very
narrow and contains the nucleus. The apical region is
expanded due to the presence of numerous mucin
granules. The latter are unstained in H&E preparations
and appear as clear vacuoles. With special mucin stains
they appear solid and stain brilliantly. Examine the
apical surface of the pseudostratified columnar
epithelium for apical specializations called cilia. Cilia
appear as small tufts or hair-like structures protruding
from the apical cell surface. When cilia are
encountered they are usually included as a prefix in the
name of the epithelium. Hence, the epithelium lining
the trachea would be termed a ciliated
pseudostratified columnar epithelium.
Examination of the epididymis with the low-
power objective reveals an organ that consists of
several profiles of small tubules. In actual fact, the
epididymis consists of one extensively coiled,
elongated tubule. An appreciation of this fact at the
start is of importance in developing three-dimensional
mental reconstructions of images from two-
dimensional images examined under the microscope.
Examine the pseudostratified columnar epithelium
lining the epididymal tubule at increased
magnification. Note that it consists of two distinct cell
types: a small basal cell and a tall columnar cell known
as the principal cell. The latter have elongate, branched
microvilli extending from their apical surface, which
are called stereocilia.
Make a sketch of the ciliated pseudostratified columnar
epithelium lining the trachea and compare it with an additional
sketch illustrating the pseudostratified columnar epithelium
lining the ductus epididymidis.
Transitional epithelium
Transitional epithelium is a stratified cuboidal type of
epithelium found only in the urinary system. Examine
this epithelium lining the interior of either the urinary
bladder or ureter. Note the thickness of this
epithelium and the large dome-shaped cells at the
luminal surface. The latter may be binucleate.
Make a sketch of transitional epithelium from the lining of the
ureter.
Specializations associated with the cell membrane
of epithelial cells
Microvilli
Closely re-examine the apices of cells forming the
simple columnar epithelium lining the small intestine.
Note the striated (microvillus) border found at this
location. Next, find and examine the proximal
convoluted tubule of the kidney. This tubule is found
only in the renal cortex (the outer region of the
kidney), is the longest tubule in the cortex (therefore
exhibits the most numerous tubular profiles) and is the
most granular and darkly stained tubule. The apices of
cells forming the proximal tubule exhibit numerous
microvilli closely packed together to form the brush
border of light microscopy.
11
Re-examine the stereocilia on the apices of the
epithelial cells lining the tubule of the epididymis.
Stereocilia are a thin, highly branched form of
microvillus.
Cilia
Re-examine the ciliated pseudostratified columnar
epithelium lining the trachea. Note that individual cilia
can be seen. If the preparation is good (particularly if
the embedding medium is a plastic resin of some type)
numerous basal bodies can be visualized in the apical
cytoplasm immediately beneath the cilia and appear as
a dense beaded line. Examine the ciliated simple
columnar epithelium lining the oviduct for the same
features.
Make sketches comparing the apical specializations found on the
epithelial surfaces examined above. In addition, examine several
textbook illustrations of each apical specialization for their
ultrastructural features.
Basal striations
Two organs that contain cells that show excellent
examples of this specialization of the cell membrane
are the distal convoluted tubules of the kidney cortex
and the striated ducts of the submandibular gland. Re-
examine the cortical region of the kidney with the low-
power objective for light-staining tubules. When
examined at higher magnification note that these
lighter-staining epithelial cells show distinct nuclear
profiles and lack the microvillus border observed in
cells forming the proximal convoluted tubules.
Careful examination of the basal cytoplasm of cells
forming the distal tubule will reveal faint striations.
The intensity of light in the microscope may have to
be decreased and/or the condenser lowered to make
the basal striations more visible. With special staining
techniques (iron hematoxylin - which actually
demonstrates mitochondria) the basal striations are
dramatic. Basal striations represent a complex
infolding of the basolateral cell membrane plus a
parallel arrangement of associated mitochondria.
Examine a section of submandibular gland for a
smaller caliber duct within the lobules of the gland.
The striated ducts are intralobular ducts and appear as
numerous circular profiles with wide lumina within the
glandular tissue. The epithelium lining these ducts is
simple cuboidal to columnar in type, is light staining
and agranular. Close observation of the basal
cytoplasm using the same conditions as when
examining the distal convoluted tubule of the kidney
will demonstrate faint basal striations within cells
making up the striated ducts. Note: these ducts were
so named because of the basal striations. Examine a
textbook electron micrograph of cells from the distal
convoluted tubule of the kidney for basolateral
infoldings. Compare these features with those seen
with the light microscope.
Make a sketch illustrating this morphological feature.
Table 1. Location of epithelia.
Type of Epithelium Location Specialization
Simple squamous Endothelium, mesothelium, thin segment of loop of Henle, rete
testis, pulmonary alveoli, parietal layer of Bowman’s capsule
Simple cuboidal Thyroid, choroid plexus, ducts of many glands, lens epithelial
cells, covering surface of ovary, corneal endothelium
Surface lining epithelium of stomach, gallbladder, ducts of
several glands
Surface lining epithelium of small and large intestines Striated border
Proximal convoluted tubule of kidney Brush border
Distal convoluted tubule of kidney Basal striations
Simple columnar
Oviducts, uterus, small bronchi and bronchioles Cilia
Trachea, major bronchi, eustachian tube Cilia
Large excretory ducts of glands, portions of male urethra
Pseudostratified
columnar Epididymis Stereocilia
Esophagus, epiglottis, corneal epithelium, vaginaStratified squamous Epidermis of skin Keratin
Stratified cuboidal Ducts of sweat glands, large ducts of salivary glands
Stratified columnar Large ducts of glands, cavernous urethra
Transitional Restricted to urinary system: renal calyces to urethra
12
Glandular forms of epithelium
Glands are comprised of epithelial cells specialized to
synthesize and secrete a product of some type. A
variety of different criteria can be used in the
classification of glands. The simplest classification
scheme is to divide the glands into endocrine
(secretion into the lymph/vascular system) and
exocrine (secretion onto an epithelial surface or into a
duct) glands. A consideration of the endocrine glands
will be presented later as a specific topic.
Exocrine glands can be classified further as to
whether they are unicellular or multicellular. A classic
example of a unicellular exocrine gland is the goblet
cell. Re-examine the epithelium lining the intestine and
trachea for goblet cells. Carefully study this cell, paying
particular attention to its overall shape, position of the
nucleus, and the apical accumulation of secretory
(mucin) granules. Recall that the mucin granules,
although unstained with the H&E preparation, can
usually be visualized by lowering the intensity of the
light and by lowering the condenser.
The majority of glands are multicellular
exocrine glands. Multicellular glands can assume a
wide variety of morphologies. They may occur as a
small group of secretory cells that lie wholly within an
epithelial layer, clustered about a small lumen. These
are called intraepithelial glands. An example of this
glandular organization can be found in the non-
keratinized stratified squamous epithelium lining the
penile urethrae of the male reproductive system.
Examine this lining epithelium. The intraepithelial
glands (glands of Littré) appear as clusters of clear or
light-staining cells within the more darkly stained
lining epithelium. The nuclei of component secretory
cells are generally compressed to the base and the
apical portion of the cell is filled with unstained mucin
secretory granules. A somewhat similar glandular
organization is the secretory sheet. In this case, the
cells form a continuous epithelial layer. An example of
this type of multicellular gland is the gastric lining
epithelium of the stomach. This glandular form
consists of a simple columnar, mucous secreting
epithelium that secretes directly into the lumen of the
stomach. Examine this epithelial form again.
The majority of multicellular, exocrine glands
secrete into a ductal system. These are classified
according to the morphology of the ducts and how
their secretory cells are arranged to form the secretory
portion of the gland. If the duct branches the gland
is classified as compound; if the duct does not
branch the gland is classified as simple. The secretory
cells of the gland may be arranged into tubules
and/or acini (alveoli) (berry-like end pieces).
Subsequent classification depends on the shape and
configuration of the secretory unit and whether these
portions also branch. Thus, simple glands can be
classed as simple tubular, simple coiled tubular, simple
branched tubular, or simple branched acinar (alveolar).
Compound glands are subdivided into compound
tubular, compound acinar and compound tubuloacinar
(compound tubuloalveolar).
Learning objectives for glandular epithelia and
exocrine glands:
1. Be able to classify glands according to their
histologic organization, type of material secreted and
manner in which material is secreted.
Simple glands
Simple tubular glands
Examine a section of colon with the low-power
objective. Find and examine the luminal surface of this
organ and note that it is lined by a simple columnar
epithelium. Observe the large number of goblet cells.
Identify tubular invaginations that extend from this
epithelium into the underlying tissue. These are the
simple tubular glands (intestinal glands) of the colon.
The epithelium forming the walls of these glands is the
same as that lining the surface. If the plane of section
through the wall of the colon is at an oblique angle,
the glands may appear as isolated oval or circular
collections of columnar cells surrounding a tiny lumen.
If the glands are cut parallel to their long axis, the
lumen of the gland can be traced to that of the colon.
Next, examine a section of small intestine. In the small
intestine, the simple tubular intestinal glands open
between the bases of fingerlike extensions of tissue
covered by simple columnar (intestinal) epithelium
called villi. Compare the glands at this location with
those of colon. Note the numerous mitotic figures
within the epithelium forming these glands.
Draw and label a sketch of these structures, including the
location of the simple tubular intestinal glands in a section of
colon and small intestine.
Simple coiled tubular glands
Re-examine the section of skin for eccrine sweat
glands as an example of a simple coiled tubular gland.
Using the low power objective examine the deep
subcutaneous tissue far beneath the overlying
keratinized stratified squamous epithelium for these
glands. The duct is long, extending from the surface
epithelium to deep within the underlying tissue.
13
The secretory portion ends as a highly coiled structure
similar to that of a coiled snail shell. A section through
such a unit results in several circular cross-sectional
profiles. These are lined by lightly stained simple
columnar epithelial cells. The ducts are more darkly
stained and lined by stratified cuboidal epithelium.
Simple branched alveolar glands
Continue to examine the preparation of skin for
sebaceous glands. These glands are classified as simple
branched alveolar glands and are almost always
associated with hair follicles. The latter are
invaginations of the epithelium into underlying tissue
that produce and contain hair shafts. Study the
sebaceous glands carefully. Note the presence of large
flask-shaped secretory units, the alveoli. Alveoli of
sebaceous glands consist of large cells filled with small
lipid droplets, which gives them a light-staining,
vacuolated appearance. Note that two or three alveoli
drain into a single, short unbranched duct lined by
stratified squamous epithelium. The duct empties into
the lumen of an adjacent hair follicle. The overall
three-dimensional shape of these glands is similar to a
three or four leaf clover.
Sketch, label and compare a simple coiled tubular sweat gland
with a simple branched alveolar (sebaceous) gland of the skin
(integument).
Simple branched tubular glands
Study a section taken from the pyloric region of the
stomach. Identify the gastric pits. These are tubular
invaginations of the gastric surface lined by simple
columnar epithelium that extend into the underlying
tissue. Emptying into the bottoms of the gastric pits
are the pyloric glands, an example of simple branched
tubular glands. Cells forming these glands have basal
nuclei and a light/clear supranuclear cytoplasm that
contains mucin granules. Note that in this glandular
form, the duct remains unbranched (simple) and that it
is the secretory tubule that branches.
Sketch and label the subcomponents of a simple branched
tubular gland from the pyloric region of the stomach.
Compound glands
Compound tubular glands
Examine a slide of the duodenum and scan it carefully.
Find the location of numerous light-staining, secretory
tubules of the duodenal (Brunner’s) glands within the
intestinal wall. These glands are found in the tissue
beneath the bottoms of the simple tubular intestinal
glands examined earlier in other regions of the
intestinal tract. Now examine the duodenal glands at
increased magnification. The terminal portions
(secretory units) of the duodenal glands are branched,
coiled and of uniform diameter. Component cells are
light staining with basally positioned nuclei. The
branching ducts are lined by a similar appearing
epithelium to that forming the secretory units. The
ducts of the duodenal glands unite with the bottoms
of the overlying intestinal glands. Note the differences
in the lining epithelium between glands where this
transition occurs.
Sketch and label a duodenal gland including its association with
an overlying simple tubular intestinal gland.
Compound tubuloacinar (alveolar) glands
This type of gland represents the most common
glandular organization of the compound glands. As an
example, study a section of the submandibular
(submaxillary) gland with the low-power objective
identifying several important features. The gland is
organized into lobes and lobules. These glandular
subdivisions are limited by fibers of the surrounding
connective tissue. Next identify the duct system. The
ducts can be recognized by their round profiles, wide
lumina, and the light-staining cytoplasm of component
cells. The epithelial lining is usually simple cuboidal or
simple columnar although stratified forms of both
types can be found on occasion lining the very large
ducts. Note that two categories of ducts can be
recognized: intralobular ducts and interlobular ducts.
The former occurs within the lobules and in the
submandibular gland are the most numerous. The
interlobular ducts are larger and occur between
lobules. The lining epithelium is usually simple
cuboidal or simple columnar. Can basal striations be
observed with increased magnification? Because the
branched ductal system of the submandibular is well
developed, numerous profiles of the ductal system are
observed. Carefully examine the apices of cells
forming the ductal epithelium. Note the minute, dense
staining points between cell apices. These are terminal
bars. Next, examine the secretory units of the
submandibular gland and observe that two markedly
different cell types makeup the tubules and acini
(alveoli) of this gland. The most numerous are serous
cells. Serous cells are characterized by a dark staining
cytoplasm filled with numerous, distinct secretory
granules. The basally positioned nuclei usually exhibit
a round or oval profile. The less numerous cell type
making up scattered tubules is the mucous cell. These
cells are characterized by a white (clear) cytoplasm of
frothy appearance. The latter is filled with unstained
mucin granules.
14
Nuclei appear as darkly stained, compressed profiles
positioned adjacent to the basal cell membrane. Some
serous cells are organized into small units called
demilunes that cap terminal regions of the scattered
mucous tubules.
When both serous and mucous cell types make up the
secretory units of a gland, the gland is often referred to
as a mixed gland.
Sketch and label the subcomponents of a lobe from the
submandibular gland.
15
CHAPTER 3. GENERAL CONNECTIVE TISSUE
It is important to realize from the onset that the
connective tissues are classified according to the type
and arrangement of the extracellular materials
rather than features of the cellular components, as is
true of epithelium. General connective tissues are
classified as loose or dense according to whether the
extracellular materials are loosely or tightly packed.
Loose connective tissue can be subdivided further on
the basis of special constituents such as adipose (fatty)
tissue or a concentration of specific extracellular
fibers. Dense connective tissue can be subdivided
according to whether the extracellular fibers are
randomly distributed (dense irregular connective
tissue) or orderly arranged (dense regular connective
tissue).
Loose (areolar) connective tissue
Areolar connective tissue is a loosely arranged
connective tissue that is widely distributed throughout
the body. It consists of three extracellular fibers
(collagen, reticular, elastic) in a thin, almost fluid-like,
ground substance. The latter is not preserved in
routine preparations and accounts for some, but not
all, of the spacing observed between the fibrous and
cellular components. Areolar connective tissue forms
the stroma that binds organs and the components of
organs together. It forms helices about the long axes
of expandable tubular structures, such as the
gastrointestinal tract and other visceral organs, the
ducts of glands, and blood vessels.
Fibers of connective tissue
Learning objectives for connective tissue fibers:
1. Be able to identify and distinguish between the three
types of connective tissue fibers.
2. Be able to classify general connective tissues
according to the arrangement of their extracellular
fibers.
Collagen fibers are present in all connective tissues,
vary in thickness from 1 to 10 µm and are of
undefined length. In H&E preparations they stain a
pink or pink-orange color. Because of the
proteinaceous subcomponents these fibers, dependent
on the dye used in staining, they can also be stained
blue, green, yellow or red. Examine the dermis of skin
(that region underlying the keratinized stratified
squamous epithelium [epidermis]) for collagen fibers.
Note the variation in size and the wavy, homogeneous
appearance of the pink-orange staining collagen fibers.
The majority of oval, densely staining nuclei in the
field are those of associated fibroblasts that secrete
and maintain the collagen fibers. The extent of the
fibroblast cytoplasm usually cannot be seen in H&E
preparations and what is actually visualized are
fibroblast nuclei. Examine the external surface of a
medium-sized (named) vessel for collagen fibers and
fibroblasts. If available, examine a spread preparation
of loose areolar connective tissue for collagen fibers
and fibroblasts. The advantage of this type of
preparation (usually a portion of a mesentery) is that it
is not a section of tissue but rather an intact tissue,
which is thin enough to allow the transmission of
light. Examine the fibroblasts carefully, first noting
their oval-shaped nuclei and then their associated
cytoplasm. In these preparations the extent of the
fibroblast cytoplasm can often be traced for
considerable distances. Note that the fibroblasts lie
immediately adjacent to, or on, collagen fibers, which
stain lightly (a light pink in most preparations). Close
observation of some fibroblast nuclei will reveal a light
appearing strip crossing the blue-stained fibroblast
nuclei. These strips are collagen fibers as seen against
the stained chromatin background of the fibroblast
nuclei. Return to a section of skin and re-examine the
dermis with low power. Note, once again, that it forms
a thick interwoven layer beneath the overlying
epithelium. At increased levels of magnification note
that the abundant thick, collagenous fibers are
interwoven to form a compact network. The dermis is
a classic example of dense irregular connective
tissue. Next, a longitudinal section of tendon or
ligament should be examined in detail. Note the
regular, precise arrangement of collagen fibers into
bundles that run parallel to one another. Fibroblasts
are the primary cell type present and occur in rows
parallel to the bundles of collagen fibers. Fibroblast
nuclei usually are the only feature of these cells
visualized and appear elongate and densely basophilic.
Tendons and ligaments are classic examples of dense
regular connective tissue.
Sketch and label the subcomponents of a region of dermis and
tendon (or ligament). Illustrate the arrangement of collagen fibers
and their association with fibroblasts. Examine an electron
micrograph from a textbook illustrating the fact that each
collagen fiber (type I) consists of banded unit fibrils, the smallest
morphologically defined unit of collagen.
Elastic fibers appear as thin, homogeneous
strands that are smaller and of more uniform size than
collagen fibers. Usually elastic fibers cannot be
distinguished easily in routine H&E preparations and
require special stains (orcein or Verhoeff's elastic stain)
to make them visible.
16
If a spread preparation of loose areolar connective
tissue stained to demonstrate elastic fibers is available,
examine it carefully and note darkly stained, variously
sized, thin cylindrical fibers coursing across the field.
These are elastic fibers. Look for an elastic fiber that
has been broken. Because of their elastic properties,
broken fibers will form a highly undulated snarl much
like broken elastic fibers of clothing (eg. stockings). If
a specifically stained preparation is not available,
elastic fibers can be visualized to some degree using
the same morphological criteria as demonstrated by
special staining. However, in this case the elastic fibers
stain the same color as collagen fibers but are
narrower (thread-like), smooth and homogeneous in
appearance, and of more uniform diameter than
collagen fibers.
Examine a section of a named (muscular)
artery for elastic tissue. Locate the lumen of the vessel
and examine the region immediately beneath the lining
endothelium. Note the highly scalloped, homogeneous
layer of elastic tissue (the internal elastic lamina) at this
location. If the vessel is specifically stained for elastin,
move through the vessel wall and examine it for other
dark-staining elastic fibers of various sizes. Return to
the vessel interior. The internal elastic lamina is not a
fiber per se but a thick homogeneous sheet of elastin.
Now, and in the future, when additional arteries are
encountered in routine H&E preparations, examine
these vessels for the internal elastic lamina. In
routine preparations this highly scalloped appearing
membrane, although stained similar to collagen, has a
slightly different refractive index. The appearance of
the elastin can be made more visible by dropping the
condenser of the microscope. Use the known position
to locate and examine the negative image of the
internal elastic lamina using this technique. Examine
sections of any other tissue specially stained to
demonstrate elastic fibers. Examine them in both
longitudinal and transverse profiles. Note again the
smooth, homogeneous nature of these darkly stained
fibers. They often give a "copper wire-like" appearance
when seen in sections of tissue.
Reticular fibers, like elastic fibers, are not
seen in routinely prepared sections but can be
demonstrated with silver stains or by the periodic acid-
Schiff's (PAS) procedure. These are small fibers that
form delicate networks and are a major component of
the stroma that binds the cells of tissues and organs
together. The most commonly used organs to
demonstrate reticular fibers are the liver, kidney,
spleen and lymph node where these fibers are
especially prominent. With silver-stained preparations
the reticular fibers stain black. Note the fine delicate
network of fibers supporting the cellular components
(parenchyma) of these organs.
Make a sketch of reticular fibers and their association with
parenchymal elements. Compare these fibers with a sketch of
elastic fibers.
Table 2. Key histologic features of connective tissue fibers.
Fiber type
Light microscopic appearance Primary locations
Collagen fibers (type I collagen)
Coarse fibers 0.5-10.0µm in
diameter, indefinite length, stain
with protein dyes
Tendon, ligament, dermis, fascia,
capsules, sclera, bone, dentin
Reticular fibers (type III collagen)
Delicate network of fine fibers,
must be stained specifically to be
demonstrated, usually by a
reduction of silver or the periodic
acid Schiff’s (PAS) staining
reaction
Stroma of lymphatic organs, bone
marrow, glands, and adipose tissue
Elastic fibers Smooth, homogeneous fibers of
varying diameter, must be stained
specifically to demonstrate well
(orcein or Verhoeff’s stain)
Dermis, lung, arteries, organs that
expand
Cells of connective tissue
General connective tissue may contain a wide variety
of cell types. Some are indigenous (residents) of
connective tissues; others are transients and migrate to
and from the general connective tissue from the
vasculature.
Learning objectives for connective tissue cells:
1. Be able to distinguish and identify the following cell
types (both indigenous and transient cells) found
within connective tissues: fibroblasts, macrophages,
plasma cells, fat cells, mast cells, neutrophils,
eosinophils and lymphocytes.
17
Indigenous cells
Fibroblasts are the most common of the connective
tissue cell types. They are large, spindle-shaped cells
with elliptical nuclei. The boundaries of the cell are not
seen in most routine preparations and the morphology
and staining intensity of the nuclei vary with the state
of activity. Active fibroblasts exhibit plump, light-
staining nuclei; nuclei of inactive fibroblasts appear
narrow and densely stained. Re-examine preparations
of dermis, tendon (ligament) and areolar connective
tissue and compare fibroblast nuclei.
Macrophages are abundant in general areolar
connective tissue. They are commonly described as
irregularly shaped cells with blunt cytoplasmic
processes and ovoid or indented nuclei that are smaller
and stain more deeply than those of fibroblasts. In
actual fact, unless macrophages show evidence of
phagocytosis, they are difficult to distinguish from
fibroblasts. If special preparations are available,
utilizing tissues from animals injected with India ink or
trypan blue, examine the areolar connective tissue or
liver preparations for cells that have phagocytized the
materials injected. These will be macrophages. One
location in which to examine macrophages in a
"natural" setting is the center (medulla) of lymph
nodes. Examine a routinely prepared lymph node
under low power. Note that its central region is lighter
staining and consists of anastomosing cords of cells
separated by wide spaces. Examine the cords of cells
and the adjacent spaces carefully for large rounded
cells with brown-gold colored particulate material
within their cytoplasm. These are macrophages.
Continue to look carefully within the interior
of the medullary cords of the lymph node and note
numerous plasma cells. These cells appear somewhat
"pear-shaped" with small eccentrically placed nuclei in
which the heterochromatin is arranged into coarse
blocks forming a clock face pattern. The cytoplasm
is basophilic and a weakly stained or light area of
cytoplasm often appears adjacent to the nucleus on the
side facing the greatest amount of cytoplasm. This
light-staining area is referred to as a negative Golgi
image. If available, examine a section of lactating
breast. The connective tissue surrounding the
secretory units of this gland often contains numerous
plasma cells. Plasma cells also are present in large
numbers in the connective tissue (lamina propria) of
the intestinal tract. Examine the connective tissue that
lies between adjacent intestinal glands for both plasma
cells and small lymphocytes. The latter show a round,
dense nucleus and only a scant rim of cytoplasm.
Fat cells are specialized for synthesis and
storage of lipid. Individual fat cells may be
encountered throughout the loose areolar connective
tissue or may accumulate in large numbers to form fat
(adipose) tissue. In routine sections, fat cells appear
large, round and empty due to the loss of a stored
central lipid droplet during tissue preparation. The
remaining cytoplasm appears only as a thin rim around
a large empty central space and if the nucleus is
encountered in these large cells, it lies flattened on one
side of the cell. Groups of fat cells have the
appearance of chicken wire or a honeycomb. Fat cells
are abundant in the hypodermis of skin - that region
of tissue lying beneath the dermis. Fat cells are also
common in the loose areolar connective tissue around
the perimeter of a lymph node. If the lymph node
preparation has been stained to demonstrate reticular
fibers, examine the delicate network of reticular fibers
enveloping each fat cell.
Mast cells are large, ovoid cells 20 - 30 µm in
diameter with large granules that fill the cytoplasm.
The nucleus is oval or round in shape and centrally
located. Mast cells are present in variable numbers in
loose connective tissue and often accumulate along
small blood vessels. Examine a mesentery or areolar
spread preparation for these large granulated cells. The
granules can be stained different colors depending on
the dye used. In tissues embedded in a plastic resin
and stained with H&E, mast cell granules stain a light
red color. If sections of this type are available, examine
sections of the stomach and intestinal tract. In either
organ the outer supporting muscular wall and adjacent
connective tissue components should be examined for
mast cells. They will appear as oval-shaped cells or
cytoplasmic fragments of cells packed with coarse red-
orange granules.
Transient cells derived from blood
Variable numbers of leukocytes constantly migrate
into the connective tissues from the blood to carry out
their specialized functions. Neutrophils are one type
of leukocyte characterized by a multilobed nucleus and
a faintly pink-staining cytoplasm. They are generally
round in shape. Most often, the multilobed nucleus is
the only prominent feature recognized when these
cells are encountered in a section of general
connective tissue. Eosinophil leukocytes also exhibit
multilobed nuclei and are characterized by bright red
(eosinophilic)-staining granules within the cytoplasm.
Lymphocytes are smaller leukocytes (5 - 7 µm in
diameter) characterized by a central, round nucleus
surrounded by a thin rim of cytoplasm. In tissue,
groups of lymphocytes appear only as collections of
round, dark staining nuclei. Examine the outer, more
darkly stained region (cortex), of a lymph node and
thymus for these small darkly stained leukocytes.
Examine the lighter-staining central region (medulla)
of the thymus for mast cells and eosinophils.
18
Sketch and compare the various cell types encountered in general
connective tissue.
Questions:
In sections of plastic embedded material stained with
H&E, the cytoplasmic granules of both mast cells and
eosinophil leukocytes stain red. How can these two
cell types be distinguished from one another? Clue:
consider their size and the nuclear profiles.
As plasma cells and lymphocytes are closely related,
how do they differ morphologically?
Table 3. Key cytologic features of cells found in general areolar connective tissue.
Cell types Nuclear characteristics Cytoplasmic characteristics
Indigenous cells
Fibroblasts Oval, centrally placed, staining
intensity variable depending on
activity<