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Preface
With this 15th edition, funqueira'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 ofbiochemistry,
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 ofconcepts 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 Thbles 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
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 ofnew 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 ofmaintaining 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 &
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 ]unqueira's
Basic Histology. Finally, I am pleased to acknowledge the help
and collegiality provided by the staffofMcGraw-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
This page inte7tionally ldt blank
CHAPTER
Histology &Its
Methods of Study
PREPARATION OF TISSUES FOR STUDY 1 AUTORADIOGRAPHY 9
Fixation 1 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

1
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
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 1-1m. 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. ForTEM, sections less than
1 1-1m 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.

Embedding II 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 f1ln thickness for light microscopy,
but electron microscopy requires sections less than I fUll thick.
One micrometer (1 flln) equals 1/1000 of a millimeter (mm)
or 10-6 m. Other spatial units commonly used in microscopy
are the nanometer (1 nm = 0.00lf1ln = 10~ mm =10-9 m) and
angstrom (1 A= 0.1 nm or 10--4flln). 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 cryostilt 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.
Preparation of Tissues for Study 3
Staining
n
Most cells and extracellular material are completely color-
less, and to be studied microscopically tissue sections must ,.
:I:
be stained (dyed). Methods of staining have been devised that -a
....
make various tissue components not only conspicuous but also m
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
ofmacromolecules in tissues. Cell components, such as nucleic
..
::a
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, aldan 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 acld-Schlff (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.
4 CHAPTER 1 • Histology & Its Methods of Study
FIGURE 1-2 Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining.
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 2~ 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
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 fllll• which can permit
clear images magnified 1000-1500 times. Objects smaller or
thinner than 0.2 fllll (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 fllll· 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

FIGURE 1-3 Componentsandlightpathofa
bright-field microscope.
Stand
I
translation
mechanism
Photograph of a bright-field light microscope showing its
mechanical components and the pathway of light from the
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
• The condenser collects and focuses a cone of light that
illuminates the tissue slide on the stage.
• Objective lenses enlarge and project the illuminated
image of the object toward the eyepiece. Interchangeable
objectives with different magnifications routinely used in
histology include X4 for observing a large area (field) of the
tissue at low magnification; Xl 0 for medium magnification
of a smaller field; and X40 for high magnification of more
detailed areas.
• The two eyepieces or oculars magnify this image another
Xl 0 and project it to the viewer, yielding a total magnifica-
tion of X40, Xl 00, or X400.
(Used with permission from Nikon Instruments.)
stained tissue preparation to high-resolution digital images
and permits study of tissues using a computer or other digi-
tal device, without an actual stained slide or a microscope. In
this technique, regions of a glass-mounted specimen are cap-
tured digitally in a grid-like pattern at multiple magnifications
using a specialized slide-scanning microscope and saved as
thousands of consecutive image files. Software then converts
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
cost and ease of use, virtual microscopy is rapidly replacing
light microscopes and collections of glass slides in histology
laboratories for students.
Light Microscopy 5
Fluorescence Microscopy
n
When certain cellular substances are irradiated by light of a :I:
proper wavelength, they emit light with a longer wavelength- >
~
a phenomenon called fluorescence. In fluorescence
-1
microscopy, tissue sections are usually irradiated with ultra- m
violet (UV) light and the emission is in the visible portion of
the spectrum. The fluorescent substances appear bright on
a dark background. For fluorescent microscopy, the instru-
ment has a source of UV or other light and filters that select
..
:u
rays of different wavelengths emitted by the substances to be
visualized.
Fluorescent compounds with affinity for specific cell
macromolecules may be used as fluorescent stains. Acridine
orange, which binds both DNA and RNA, is an example.
When observed in the fluorescence microscope, these nucleic
acids emit slightly different fluorescence, allowing them to be
localized separately in cells (Figure 1-4a). Other compounds,
such as DAPI and Hoechst, stain specifically bind DNA and
are used to stain cell nuclei, emitting a characteristic blue fluo-
rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as •
fluorescein to molecules that will specifically bind to certain I"'"
IS'
cellular components and thus allow the identification of these ....:::r
structures under the microscope (Figure 1-4b). Antibodies s::
;:;·
labeled with fluorescent compounds are extremely important
in immunohistologic staining. (See the section Visualizing a
1ft
Specific Molecules.) s
~
Phase-Contrast Microscopy
Unstained cells and tissue sections, which are usually trans-
parent and colorless, can be studied with these modified
light microscopes. Cellular detail is normally difficult to see
in unstained tissues because all parts of the specimen have
roughly similar optical densities. Phase-contrast micros-
copy, however, uses a lens system that produces visible images
from transparent objects and, importantly; can be used with
living, cultured cells (Figure 1-5).
Phase-contrast microscopy is based on the principle that
light changes its speed when passing through cellular and
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.
Because they allow the examination of cells without fixation or
staining, phase-contrast microscopes are prominent tools in
all cell culture laboratories. A modification of phase-contrast
microscopy is differential interference contrast micros-
copy with Nomarski optics, which produces an image of liv-
ing cells with a more apparent three-dimensional (3D) aspect
(Figure 1-Sc).
Confocal Microscopy
With a regular bright-field microscope, the beam of light
is relatively large and fills the specimen. Stray (excess) light
reduces contrast within the image and compromises the
6 CHAPTER 1 • Histology & Its Methods of Study
FIGURE 1-4 Appearance of cells with fluorescent microscopy.
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.
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
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
XSOO)
(Figure 7-4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
Bloomington.)
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.)
 Light Microscopy 7

such optical sections at a series of focal planes through the

FIGURE 1-6 Principle of confocal microscopy. n
specimen allows them to be digitally reconstructed into a
:I:
3D image.
>
~

Polarizing Microscopy -1
m

..
Polarizing microscopy allows the recognition of stained or :u
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits
vibrating in only one direction. If a second filter is placed in
the microscope above the first one, with its main axis per-
Detector
I 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
of vibration of polarized light is called birefringence and is

--Beam
splitter FIGURE 1-7 Tissue appearance with bright-field
and polarizing microscopy.
•
I"'"
IS'
....:::r
i:
;:;·
a
1ft

s
~

Although a very small spot of light originating from one plane

ofthe 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-
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
Polarizing light microscopy produces an image only of material
each other in the focal plane (confocal), and unfocused light having repetitive, periodic macromolecular structure; features
does not pass through the pinhole. This greatly improves without such structure are not seen. Pieces of thin, unsec-
resolution of the object in focus and allows the localization tioned mesentery were stained with red picrosirius, orcein, and
of specimen components with much greater precision than hematoxylin, placed on slides and observed by bright-field (a)
and polarizing (b) microscopy.
with the bright-field microscope.
Confocal microscopes include a computer-driven mirror (a) With bright-field microscopy, collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
system (the beam splitter) to move the point of illumination
(b) With polarizing microscopy, only the collagen fibers are
across the specimen automatically and rapidly. Digital images
visible and these exhibit intense yellow or orange birefrin-
captured at many individual spots in a very thin plane of focus gence. (a: X40; b: X1 00)
are used to produce an "optical section'' of that plane. Creating
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.
FIGURE 1-8 Electron microscopes.
Intermediate lens--~
Projector lens----~
Image on viewing - -----4!;
screen
Electron detector
with ceo camera
(a) Transmission 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.
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
Cathode------~
(b) Scanning electron microscope
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 30, shadowed quality. The inside of organs or cells
can be analyzed after sectioning to expose their internal surfaces.

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 Microscopicautoradiography.
Auto radiographs 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 3 H-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.
Autoradiography 9
layer of heavy metal (often gold) that reflects electrons in a
beam scanning the specimen. The reflected electrons are cap- n
:I:
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 -1
m
appears to be illuminated in the same way that large objects are
seen with highlights and shadows caused by light.
>AUTORADIOGRAPHY
..
:u
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
,.c•
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia- ..
0
ii'l
tion produce small black grains of metallic silver, which under a.
either the light microscope or TEM indicate the locations of ~·
radiolabeled macromolecules in the tissue (Figure 1-9). ii'l
Much histological information becomes available by 'a
:::r
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
(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. (XlSOO)
(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 7-9b, used with permission from Drs Ticiano G. Lima and
A. Antonio Haddad, School of Medicine, Ribeircio Preto, Brazil.)
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 ti rst cell lines,
called HeLa cells, which are still used in research on cellular
structure and function throughout the world.
> 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 aldan blue
reactions for polysaccharides (to detect glycogenosis and
mucopolysaccharidosls), 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.

FIGURE 1-10 Enzyme histochemistry.
(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 7-7 Ob, used with permission from Dr Eduardo
Katchburian, Department of Morphology, Federal University of
Sao Paulo, Brazil.)
Visualizing Specific Molecules 11
Examples of molecules that interact specifically with
n
other molecules include the following:
:I:
• Phalloidin, a compound extracted from mushroom, >
~
Amanita phalloides, interacts strongly with the actin pro-
-1
tein of microfilaments. m
• Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fe region of antibody molecules, and
can therefore be used to localize naturally occurring or
applied antibodies bound to cell structures.
..
:u
• 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 s
1ft
c
histochemistry. !.
The body's immune cells interact with and produce anti- iii'
:;·
bodies against other macromolecules-called antigens-that 1&2
are recognized as "foreign;' not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
"'n
'a
ID
noglobulin family of glycoproteins and are secreted by lym- 3i
n
phocytes. These molecules normally bind specifically to their s::
0
provoking antigens and help eliminate them.
Widely applied for both research and diagnostic pur-
ifc
poses, every immunohistochemical technique requires an iD
1ft
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 ofbinding 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
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 lmmunocytochemistrytechniques.
-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
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 oflabeled 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
andTEM.
>> 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.
 Visualizing Specific Molecules 13

FIGURE 1-12 Cells and tissues stained by immunohistochemistry. n

:I:
>
~

-1
m

..
:u

the cytoplasm. Primary antibodies against the filament pro-

tein desmin and fluorescein isothiocyanate (FITC)-Iabeled
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
s
1ft

the enzyme lysozyme. The secondary antibody labeled with

c
!.
peroxidase was then applied and the localized brown color iii'
produced histochemically with the peroxidase substrate :;·
1&2
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
"'n
'a
ID

hematoxylin. (Xl 00) 3i

b
Immunocytochemical methods to localize specific proteins can
be applied to either light microscopic orTEM preparations using
a variety of labels.
(a) A single cultured uterine cell stained fluorescently to reveal
a meshwork of intermediate filaments (green) throughout
n
(c) A section of pancreatic cells in a TEM preparation incubated s::
0
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin- ifc
ity toward antibody molecules and the resulting image reveals iD
1ft
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.
(XSOOO)
(Figure 7-7 2c, 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
Another random document with
no related content on Scribd:
Prima però che mettiamo il piede nel pompejano sobborgo,
demandiamo a’ libri antichi le costumanze che precedevano la
tumulazione: la visita a’ sepolcri non sarà che il complemento del
nostro tema. E avanti tutto, ricostruendo colla nostra fantasia sui
ruderi d’una di queste case l’intero edificio e animandolo de’ suoi
antichi abitatori, conduciamoci al cubiculum, dove sul ricchissimo
letto giace il pater familias in preda a morbo letale.
Sul monopodium marmoreo, o tavola di un sol piede, di cui gli scavi
offersero un esemplare, stanno i vasi e le ampolle del seplasarius, o
farmacista e che il medico ha prescritte; ma l’aspetto dei congiunti
accusa che poco oramai si attenda da que’ farmachi studiati.
Quando il medico o la natura avvertivano finalmente che all’infermo
più non restava speranza di vita, e che era prossimo al suo estremo
fato, la famiglia e i parenti di lui gli si raccoglievano intorno al letto,
come se si trattasse di dar l’ultimo saluto a chi fosse per partire per
un lungo viaggio. Era infatti per il viaggio che non aveva ritorno. Essi
iscongiuravano altresì la morte ed impetravano da Mercurio la grazia
che volesse servire di guida all’anima che stava per entrare nella
regione de’ morti. E quando l’agonia pareva incominciata, si aveva
cura di chiudergli gli occhi, acciò non fosse egli contristato dallo
spettacolo che precede la morte, o perchè meno formidabile gliene
apparissero le dimostrazioni. Il figlio, o il più prossimo parente,
dandogli l’ultimo bacio, ne raccoglieva l’estremo sospiro, e tale era
un conforto che auguravansi le madri di ciò fare coi loro figli, giusto
quanto Cicerone afferma: Matresque miseræ nihil orabant nisi ut
filiorum extremum spiritum excipere sibi liceret [218]. Nè altrimenti era
in Grecia fin da più remoti tempi e ce ne persuade Omero
nell’Odissea, dove Agamennone si lagna di Clitennestra:

. . . . al marito,
Che fra l’ombre scendea, non chiuse il ciglio
E non compose colle dita il labbro [219]:

e Virgilio attesta dell’uso recato in Italia, quando mette sulla bocca

della madre d’Eurialo il lamento:
 . . . . nec te, tua funera, mater,
Produxi, pressive oculos... [220]

uso continuatosi sempre dipoi; onde Lucano nella Pharsalia disse

pure:

. . . . tacito tantum petit oscula vultu,

Invitatque patris claudenda ad lumina dextram [221].

Reso il quale, per tre volte, a distinti intervalli, si chiamava ad alta

voce il morto, ciò che dicevasi conclamatio; e conclamatum est,
significava adunque che una cosa più non esisteva.
Ma per apprezzare convenientemente questa cerimonia e le
numerose altre che praticavansi in occasione di morte presso i
Romani, gioverà vedere dapprima quali fossero presso di essi le
credenze sull’anima e sulla morte.
E mi affretto a mettere in sodo come il non omnis moriar non fosse
già un principio suggerito al poeta dalla coscienza della immortalità
delle sue concezioni intellettuali, sibbene la radicata credenza che si
aveva in una seconda vita, dopo questa terrena. Non fu quindi il
portato d’una dottrina speculativa o filosofica qualunque, ma fu
veramente una credenza questa di lunga mano anteriore
all’almanaccar de’ filosofi, anzi precorritrice d’assai alla loro
esistenza; di modo che la morte venisse considerata come una
semplice mutazione della vita.
Dove poi versasse questa seconda esistenza al di là della tomba,
variò la credenza.
Secondo le più antiche opinioni de’ Greci e degli Italioti, che per lo
più divisero costumi e credenze insieme, come veramente usciti d’un
solo ceppo, per testimonianza di Cicerone: sub terra censebant
reliquam vitam agi mortuorum [222], ed anzi pensavano restasse
l’anima tuttavia consociata al corpo; onde così spiegar ci possiamo
l’espression di Virgilio ne’ funerali di Polidoro:
 animamque sepulcro
Condimus, et magno supremum voce ciemus [223].

E l’iscrizione che apponevasi al sepolcro diceva infatti: hic jacet, qui

giace, qui posa il tale, non già solo la spoglia del tale, dicitura che,
malgrado le ben diverse credenze, pur a’ dì nostri è pervenuta e
l’usiamo pure nei nostri ipogei. Era pertanto ragione che si curasse
allora di chiudere nel sepolcro dallato al cadavere gli oggetti di cui
reputavasi potesse sentire necessità e si spargesse al di sopra vino,
latte e miele e si immolassero vittime, come vedremo più avanti, allo
scopo di soddisfargliene la fame e la sete, ed anche a quello che
avesse il defunto a valersi nella tomba di quel che sulla terra
godeva.
Dopo ciò, non è più lecito credere che negli spiriti delle popolazioni
greco-italiche avesse potuto attecchire l’idea della metempsicosi
ossia della trasmigrazione dell’anima da un corpo all’altro. Nè di
meglio credevasi che l’anima, lasciando il corpo, volasse al cielo;
perocchè cotale credenza non trovò seguaci che assai più tardi,
tutt’al più reputandosi che il soggiorno celeste convenisse, come
straordinaria ricompensa a certi eroi, o benefattori della umanità.
Le più antiche generazioni credevano adunque unicamente che
l’anima non si separasse dal corpo, che rimanesse fissa a quella
parte di suolo, dove erano sepolte le sue ossa; che non dovesse
rendere conto alcuno di sua vita anteriore, che non avesse ad
attendere nè ricompensa, nè punizione.
Comunque più innanzi s’allargasse la fede e si credesse nel Tartaro,
e ne’ Campi Elisi, come luoghi di punizione il primo e di premio i
secondi per i fatti della vita terrena, i riti funerarii che mi faccio ora ad
esporre risentirono sempre delle primitive credenze, le quali
sebbene ci possano sembrare viete e perfino ridicole, secondo
giustamente osserva Fustel de Coulanges, hanno tuttavia esercitato
l’impero sull’uomo durante gran numero di generazioni: esse hanno
governato le anime, rette le società, e la più parte perfino delle
istituzioni domestiche e sociali degli antichi sono prevenute da
questa sorgente [224].
Ma ho ricordato ora gli Elisii e il Tartaro: debbo darne alcuna
nozione, perchè, come dissi, la credenza in essi divenne poi
generale e costituì il mistero pagano d’oltretomba.
Secondo i Greci, i Campi Elisi o l’Elisio era la quarta divisione
dell’inferno; secondo i Romani invece era la settima.
Vi regnava, per quanto ne testimoniò Pindaro ne’ suoi inni immortali,
eterna la primavera, vi alitavano zefiri profumati, vi sfolgoravano sole
e astri perpetuamente, vi crescevano fiori e frutta, v’era bandita la
vecchiaja, ignoti i mali, senza fine la vita e l’onda di Lete, che
tutt’all’intorno circondava quel beato soggiorno, procurava l’obblio
delle dolorose memorie del passato. Lo stesso Pindaro ne fa re
dell’Eliso Saturno: altri lo volle retto dalle leggi di Radamanto.
Diverse pure le opinioni sulla località di esso: Omero ed Esiodo lo
collocarono nel centro della terra e sulle rive dell’Oceano.
L’inferno, o Tartaro, era pei Greci un luogo sotterraneo, ove
scendevano le anime dopo la morte per esservi giudicate da
Minosse, Eaco e Radamanto, e Plutone vi regnava sovrano. Aveva
vari spartimenti: l’Erebo ove stava il palazzo della Notte, quello del
Sonno e dei Sogni, il soggiorno di Cerbero, cane ringhioso dalle tre
gole, delle Furie e della Morte; l’inferno delle anime prave punite nel
fuoco o nel ghiaccio; il Tartaro propriamente detto, dove i nuovi Numi
avevano precipitato gli antichi, i Ciclopi, i giganti e i titani.
I Romani lo dividevano in sette scomparti: nel primo soggiornano i
bambini; nel secondo gli innocenti stati condannati a morte; nel terzo
i suicidi; nel quarto gli amanti spergiuri o sfortunati; nel quinto gli eroi
che si macchiarono di crudeltà; nel sesto, detto più propriamente il
Tartaro, stavano i martiri; nel settimo i Campi Elisii che già vedemmo
essere luogo di riposo e di beatitudine pei giusti.
Facile è qui constatare come la dottrina pur del Cristianesimo
ammettesse suppergiù, con quasi identità di nome e di destinazione,
la credenza dell’Eliso e dell’Inferno, e il Cattolicesimo vi aggiungesse
un terzo luogo di depurazione, cioè il Purgatorio, ammettendo in
certo qual modo una tal qual legge di progresso, secondo anche la
dottrina spiritica moderna, che cioè permettesse alle anime non al
tutto superiori ed elette di espiare le colpe non gravi, prima d’essere
ammesse nella sede dei santi.
È poi curioso osservare da ultimo, intorno a tale argomento, come i
tre più grandi poeti dell’Umanità, Omero, Virgilio e Dante, l’abbiano
consacrato nelle loro immortali epopee: perocchè il primo, nel canto
undecimo dell’Odissea, guidasse Ulisse ne’ luoghi inferni a trovarvi i
campioni della guerra trojana: il secondo vi conducesse, nel canto
sesto dell’Eneide, il suo eroe, dove appunto divise tutti quegli
scompartimenti che ho più sopra accennati, così lasciandovi
memoria di tutte le antiche dottrine circa il soggetto; e l’Alighieri poi,
della credenza dell’Inferno, del Purgatorio e del Paradiso, costituisse
anzi tutta la macchina della sua Divina Commedia, nella quale agli
scomparti pagani sostituisce di suo capo le bolge ed i gironi, dove
pure colloca, secondo il maggiore o minor merito, la maggiore o
minore pravità, gli spiriti de’ suoi personaggi, tra’ quali con nova
fantasia vede taluni degli uomini del suo tempo ancor viventi.
I morti poi ebbero sempre tra’ Romani una religione: il culto che loro
si prestava divenne anzi così obbligatorio, che nelle XII Tavole i
relativi diritti degli Dei Mani, — che con questo nome chiamavano
generalmente i morti, — vennero positivamente sanciti, secondo il
testo conservato di questa particolare legge da Cicerone: Deorum
Manium jura sancta sunto; hos leto datos divos habento: sumptum in
illos, luctumque minunto [225]. Il medesimo Cicerone, nel Libro De
Legibus, rammentò come fosse stato volere de’ maggiori che gli
uomini che avevano lasciato questa vita, fossero annoverati tra gli
Dei [226].
L’uomo ch’era stato del suo vivente tristo, morto era egualmente un
dio: solo serbava nella seconda vita le malvagie tendenze della
prima.
Se non che Apulejo lasciò detto che quando i Mani fossero malevoli,
si dovessero chiamare larvæ, e quando benevoli, lares; perocchè
infatti Manes, Genii e Lares si dicessero promiscuamente. Udiam lui
stesso: Manes animæ dicuntur melioris meriti, quæ in corpore nostro
Genii dicuntur; corpori renuntiantes, Lemures; cum domos
incursionibus infestarent, Larvæ; contra ei faventes essent, Lares
familiares [227].
Questa religione de’ morti, scrive il dotto Fustel, che ho già citato,
sembra essere stata la più antica in questa razza d’uomini. Prima di
concepire e d’adorare Indra o Zeus, l’uomo adorò i morti: ebbe paura
di essi e indirizzò loro preghiere. Sembra così che il sentimento
religioso abbia di là avuto la sua origine. È forse alla vista della
morte che l’uomo ebbe per la prima volta l’idea del soprannaturale e
che volle sperare al di là di quel che vedeva. La morte fu il primo
mistero: ella mise l’uomo sulla via degli altri misteri: essa sollevò il
suo pensiero dal visibile all’invisibile, dal passeggiero all’eterno,
dall’umano al divino.
Pur Ugo Foscolo ne’ suoi Sepolcri fe’ rimontare il culto de’ morti allo
istituirsi del contratto sociale:

Dal dì che nozze, tribunali ed are

Diero alle umane belve esser pietose
Di sè stesse e d’altrui, toglieano i vivi
All’etere maligno ed alle fere
I miserandi avanzi che Natura
Con vece eterna a sensi altri destina [228].

Era forse anche per questo culto che gli Dei Mani si chiamassero Dii
patrii ed anche Dii sacri, come si veggono così, indicati sovra alcuni
monumenti. — Più avanti toccherò di feste ed onoranze istituite per
gli Dei Mani.
Come facciamo oggidì che denunziamo al Municipio l’avvenimento
d’una morte, e trattiamo della spesa de’ funerali, allora veniva
denunciato il decesso al tempio della dea Libitina, istituito da Numa,
nel quale si custodivano tutti gli apparati ed addobbi richiesti per
mettere in ordine un funerale e quivi col libitinario, o intraprenditore
delle pompe funebri, convengasi sull’indole di quella che ricercavasi
e sulla spesa.
Il libitinario spacciava alla casa del morto i suoi schiavi, detti
pollinctores, dal polline, dice Servio, o fior di farina onde lievemente
spargevasi la faccia del defunto, dopo che il corpo fosse stato dalle
donne con acqua calda lavato. — Tale costume di preparare,
imbiancandolo, il viso agli estinti si conserva tuttavia in Rumenia,
dove lo portarono i Romani antichi, che vi lasciarono indelebili tracce
di loro soggiorno e colonizzazione in altre molte consuetudini, nel
linguaggio e perfino nella denominazione del paese, che fino a’ dì
nostri vanta con noi comune le origini. — Quindi i medesimi
pollinctores l’ungevano e imbalsamavano con appositi aromi:

. . . corpusque lavant frigentis et ungunt [229].

Ciò eseguito, lo si rivestiva dell’abito ch’era solito portar vivo, colle

insegne che s’era meritato. Così il semplice cittadino d’una toga
bianca, il magistrato della pretesta, i censori della porpora; d’una
semplice tonaca invece gli abitanti della campagna e i plebei. Gli si
poneva in bocca un triente, cioè la terza parte di un’asse, la moneta
di rame corrispondente a due centesimi di lira italiana con cui
intendevasi pagare Caronte,

Il nocchier della livida palude,

pel tragitto di essa, pur descritto nella Cantica dell’Inferno da Dante,

e come il rammenta Giovenale:

. . . . at ille
Jam sedet in ripa, tetrumque novicius horret
Porthmea, nec sperat cænosi gurgitis alnum
Infelix, nec habet quem porrigat ore trientem [230].

Le leggi delle XII Tavole vietando di seppellire l’oro nelle tombe, —

osserva giustamente quel dotto scrittore e orientalista che è il prof.
Angelo De-Gubernatis, confermano soltanto la esistenza dell’uso
nell’antica Roma [231].
Coronandogli di fiori la testa, lo si deponeva su d’un alto letto,
d’avorio, se ricco, e coperto di preziose stofe, nel vestibolo, co’ piedi
rivolti verso l’uscita di casa, quasi ad indicarne la partenza. Persio
così ricorda sommariamente, nella satira III queste funerali
cerimonie:

Hinc tuba, candelæ; tandemque beatulus alto

Compositus alto lecto, crassisque lutatus amomis,
In portam rigidos calces extendit [232].

Quest’uso di collocare i cadaveri che si dovevano trasportare, co’

piedi vôlti all’uscita della casa, nota a questo passo Vincenzo Monti,
era antichissimo. Omero ne fa menzione nel XIX canto dell’Iliade,
ove Achille addolorato per l’estinto amico (Patroclo), così parla:

D’acuto acciar trafitto egli mi giace

Nella tenda co’ piè vôlti all’uscita.

Davanti la porta si piantava il funerale cipresso, l’albero consacrato a

Plutone, perocchè esso una volta tagliato più non ripulluli, secondo
lasciò ricordato Plinio il vecchio. La presenza del cipresso era indizio
di lutto patrizio, come avvertì Lucano nel seguente verso:

Et non plebejos luctus testata cupressus [233].

Di tal guisa restava avvertito il pontefice di tenersi lontano da quella

casa, da cui sarebbe stato polluto; così evitavanla coloro che
disponevansi a compiere alcun sagrificio, perocchè quell’impuro
contatto non avrebbe più loro concesso d’accostarsi agli altari. Così
all’eguale intento, e per tutto il tempo che duravano le funebri
cerimonie, si solevano velare le immagini degli Dei.
Per sette giorni lasciavasi esposto il cadavere, acciò si avesse tutto
l’agio di riprendere i sensi dove un letargo avesse simulato la morte,
e vegliava a studio di esso uno schiavo della casa e durante un tal
tempo facevansi da que’ della famiglia le maggiori dimostrazioni di
dolore:

. . . it clamor ad alta
Atria; concussam bacchatur fama per urbem;
Lamentis, gemituque et fœmineo ululatu
Tecta fremunt; resonat magnis plangoribus aether [234].

L’ottavo giorno un pubblico banditore, percorrendo le vie principali

adiacenti alla casa mortuaria, convocava il popolo per celebrare i
funerali. Terenzio ci lasciò nelle sue commedie rammentata la
formula di tale convocazione:

Quirites exsequias... quibus est commodum ire jam tempus est [235].

Siccome poi, giusta quanto superiormente dissi, le anime delle

persone buone si consideravano ricevute nel novero degli Dei
benefici, comunque d’ordine inferiore; così ci restò qualche
documento che prova come si ponesse nel feretro, presso il
cadavere, un attestato di buona condotta, rilasciato dal Pontefice,
perchè fosse agevolato il compito de’ giudici eterni. Bannier ne
riferisce un esempio in quello che fu posto accanto ad un cadavere
dal Pontefice Sesto Anicio, ch’io pur trascrivo, acciò si conosca
anche di questo curioso documento la formula. Eccolo: Ego Sextus
Anicius Pontifex testor hunc honeste vixisse: Manet ejus inveniant
requiem [236].
Funus dicevasi il funerale, a cagione che negli antichi tempi i romani
seppellissero di notte al lume di candele, o torcie, che si formavano
di funi ritorte, funalia, intrise di pece, portate dai piagnoni. Non fu che
più tardi che l’uso di seppellire di notte si restrinse alle classi più
povere, le quali non potevano sostener la spesa di splendide
esequie.
Ed oltre di tal distinzione, diverse altre erano le specie di funerali.
Publicum era quel funerale che si faceva a spesa dello Stato, come
in quest’anno in cui scrivo (1873, 29 maggio), Milano praticò a
riguardo di Alessandro Manzoni, morto il 22 dello stesso mese e
riuscì così imponente e pomposo da potersi dire per lo appunto quel
che Plinio scrisse a Romano del funerale pubblico di Virginio Rufo, il
quale, se non come Manzoni ebbe a vivere ottantotto anni, ne visse
nondimeno ottantatre, compiuti in una beatissima quiete e in non
minore venerazione, che stato console per tre volte, arrivò all’apice
degli onori privati e sopravvivendo trent’anni alla sua gloria, lesse
versi, lesse storie, scritti in suo onore e conversò in certa guisa co’
posteri: Post aliquot annos insigne, atque etiam memorabile populi
romani oculis spectaculum exhibuit publicum funus Virginii Rufi
maximi et clarissimi civis, perinde felicis [237]. Il Paravia in nota a
questa lettera di Plinio, affermò chiamarsi anche censoria questi
publici funerali, rimettendo circa alle cerimonie, al lusso ed anche
alla stravaganza di queste funebri solennità alle antichità Romane di
Adam (vol. III e IV) che ne fece la descrizione. Ma il funerale publico,
fatto a spesa dello Stato, che in quest’anno medesimo rimase più
memorabile ancora, fu quello che si celebrò in Roma nel 7 giugno
1873, per Urbano Rattazzi, il più eminente uomo di Stato che aveva
l’Italia, stato sei volte ministro di re Vittorio Emanuele, morto il 5 dello
stesso mese in Frosinone, e il cui nome, come quello di carissimo e
venerato amico, io rammenterò nelle lagrime finchè vita mi rimarrà.
Non fu pompa solo ufficiale, ma, come fu egregiamente detto, fu
vero plebiscito: poichè tutte le città vi partecipassero nel lutto, e con
essa i principi reali, i più alti dignitari, senatori e deputati, illustri
stranieri e d’ogni ordine cittadini. Splendidissimi del pari poi, ed a
spesa del Municipio, Alessandria sua patria gli rinnovò l’undici
giugno successivo, quando essa ebbe il cadavere che reclamò e che
venendo da Roma ebbe lungo il viaggio le ovazioni delle popolazioni
in mezzo alle quali passava.
Funus indictivum appellavasi quel grande funerale in cui veniva
invitato il popolo a’ ludi gladiatorii ed alle militari rassegne, che si
offerivano ad onoranza di illustre defunto; mentre tacitum o
translatitium dicevasi il funerale comune ed ordinario senza veruna
ostentazione di potenza. Funus gentilitium era poi quello nel quale si
recavano in processione le imagini de’ maggiori della medesima
prosapia, gens.
Fatta dal banditore l’ultima conclamazione, il designator, o maestro
della funebre cerimonia, assistito da’ suoi littori, o da un suo accolito,
accensus, ordinava che la processione si incamminasse per
trasferire il cadavere all’ultima dimora, tutti recando, comunque fosse
di giorno, torcie accese nelle mani, in memoria dell’antico costume.
Apriva la marcia una banda di musicanti, siticines, che suonavano la
tibia longa, o flauto funebre, accompagnando con essa un canto
lugubre in lode del trapassato, come ci spiegò Novio Marcello:
Siticines dicti sunt qui funeratos et sepultos canere soliti erant causa
honoris cantus lamentabiles [238].
Il mortoro de’ grandi e delle persone attempate, quando il publico era
stato convocato, veniva accompagnato da trombettieri, tubicines, i
quali annunciavano che il defunto non era stato tolto di vita dal ferro
o dal veleno.
Dietro i musici venivano le prefiche, præficæ, schiave del libitinario
incaricate di fare il piagnisteo; ed esse, mediante pagamento,
percuotevansi il petto, mandavano grida strazianti e strappavansi i
capelli, ostentando un dolore fierissimo che erano ben lungi dal
sentire. Così Lucilio nelle satire ci descrive la loro simulata
desolazione:

Mercede quæ
Conductæ fient alieno in funere præficæ
Multo, et capillos scindunt et clamant magis [239].

L’uso delle prefiche, comune a quasi tutte le nazioni, si protrasse

tardissimo anche fra noi. Nella diocesi di Milano vennero proibite
dall’Arcivescovo S. Carlo Borromeo. — E celebravano esse talvolta
le lodi del defunto col canto, nænia, e tal altra recitando passi de’
poeti più rinomati che avessero qualche analogia colla circostanza.
Erano così insinceri siffatti canti laudativi, che passò di poi nænia per
sinonimo di nugæ, ossia bagatelle od inezie. Il nostro Porta,
l’insuperabile poeta del nostro vernacolo, disse alla sua volta bosard
come on cartell de mort, bugiardo come un cartellone da morto, o,
come potrebbesi anche dire, al pari di un epitaffio. Guasco ricorda
che dietro le prefiche venissero altre donne: Piatrices, Sagæ,
Expiatrices, Simpulatrices, ed erano sacerdotesse che presiedevano
a’ sacrificj impetratorj per ottenere l’ingresso del defunto negli abissi,
ed espiatorj per purgarsi dai peccati [240].
Seguivano i Popi e i Victimarii: ufficio dei primi era di abbattere gli
animali più diletti al defunto padrone, come cavalli, cani, ed uccidere
uccelli alla pira funebre: dei secondi di predisporre gli arredi
necessarii all’uopo. Essi apparivano nudi fino alla cintura.
Poi quelli che portavano le insegne onorifiche del morto, come le
spoglie prese al nemico, i distintivi ed i premii conseguiti dal suo
coraggio, ogni cosa però capovolta a dimostrazione di lutto.
Portavansi pure le imagini degli avi illustri disposte per ordine
cronologico su’ carri, pilenta, le insegne delle magistrature e delle
dignità coperte da essi, e siffatto privilegio spettava pure alle donne
ne’ loro funerali, dove avessero avuto negli antenati loro taluno che
avesse sostenuto una magistratura curule.
Teneva dietro tutto ciò una schiera di mimi e l’archimimus o capo di
essi. I primi ballavano danze grottesche al suon de’ crotali, le quali
danze chiamavansi sicinnia, i cui salti regolavansi in misura co’ piedi
dattili dell’anapesto, metro simile a un dipresso al quinario nostro e
del quale eccone esempio tolto a Seneca il tragico:

Fundite fletus,
Edite planctus,
Fingite luctus,
Resonet tristi
Clamore forum, ecc. [241]

Il secondo, imitava coll’incesso e co’ gesti il costume, i modi più

spiccati e la persona del defunto, come viene attestato dallo storico
de’ Cesari, Svetonio [242].
Venivano ultimi i parenti e gli amici, spogliate le dita d’anelli e colla
barba intonsa, vestiti tutti della penula oscula, abito di rigore nelle
funerali pompe, nelle quali non era permesso portare la toga e
comprendevasi essa fra i vestimenta clausa. I figliuoli incedevano
colla testa coperta, le figlie invece a capo scoperto: queste poi, la
madre e la moglie senza ornamento, colle chiome disciolte ed in
nere gramaglie. Le donne solevano mostrare un vivo dolore,
straziandosi il seno nudo ed il volto, tanto da spicciarne il sangue, e
invocando l’amato defunto ad alta voce, come Properzio desiderava
avesse a fare per lui la bella Cinzia:

Tu vero nudum pectus lacerata sequeris.

Nec fueris nomen lassa vocare meum [243]

e ciò non a vana dimostrazione di duolo, ma perchè, secondo

spiegano i commentatori, i mani amano il latte ed il sangue.
Dopo di costoro, procedeva la bara, capulum, feretrum, lectica
funebris, come poteva venire con tutti questi nomi designata, ed era
un letto od anche una lettiga coperta da più o men ricco drappo, a
seconda della varia dignità dell’estinto e portata da’ più prossimi
parenti o dagli amici, in numero di sei o di otto, e per ciò detta anche
exaphorum, od octophorum. Talvolta sorreggevasi essa dagli schiavi
dichiarati liberi nel testamento, e tenevano allora in segno di loro
recente libertà coperto il capo. I personaggi alto locati erano portati
da dignitarj o funzionarj dello stato; la bara di Lucio Cornelio Silla
dalle Vestali, quella di Giulio Cesare dai magistrati, quella di Augusto
da’ senatori, quella di Tiberio da’ soldati, e Tacito ricorda che il
feretro di Germanico venisse portato sulle spalle de’ tribuni e de’
centurioni [244]; ma poi e più innanzi l’urna dell’imperatore Severo fu
portata per mano dei consoli medesimi.
Dietro la bara succedeva la caterva de’ clienti, degli schiavi e de’
familiari, conducendo a mano gli animali che dovevano essere
sagrificati al bruciamento del cadavere, e finalmente chiudevasi la
processione colla carrozza vuota, rheda o carpentum, del defunto e
colla turba de’ curiosi e sfaccendati che mai non mancano agli
spettacoli che si offrono gratuitamente.
Il funerale corteggio, quando trattavasi di persona illustre o ricca,
soffermavasi un tratto nel foro, dove, posto il funebre letto sulla
tribuna, un prossimo congiunto, o l’erede beneficato, pronunciava
l’orazione funebre in mezzo ai suoni lugubri di una musica mesta, ciò
che diede origine alla frase latina laudare pro rostris. Ricorda il
lettore come Svetonio, nella vita di Cesare, lasciasse memoria aver
questi alla sua volta arringato dai rostri l’orazion funebre per la sua
zia (amita) Giulia e per la moglie Cornelia, cogliendo il destro così di
vantarsi disceso per una parte da regale prosapia e per l’altra da
Venere, affine poi d’inferirne: est ergo in genere et sanctitus regum,
qui plurimum inter homines pollent, et cærimonia deorum, quorum
ipsi in potestate sunt reges [245].
Il De-Gubernatis nella sua opera sullodata degli Usi Funebri Indo
Europei [246], ricorda come Plutarco nella vita di Valerio Publicola
riferisca a questo console l’origine della istituzione delle orazioni
funebri romane. «Ebber cari i Romani — queste son le parole dello
scrittore delle Vite degli uomini Illustri — quegli onori che fece Valerio
al suo collega (Bruto) coi quali illustrar ne volle il mortorio, e
specialmente l’orazion funebre che recitò in di lui lode egli stesso, la
quale riuscì di tanta soddisfazione e fu sì grata ai Romani medesimi,
che introdotto indi venne il costume di encomiarsi dopo morte, in tal
guisa, tutti i grandi, e valentuomini dai personaggi più insigni. Questa
orazion funebre, secondo si dice, fu più antica anche di quella de’
Greci, se pure anche ciò non fu una istituzione di Solone, come
lasciò scritto il retorico Anassimene.» Dionigi d’Alicarnasso, nel libro
quinto delle sue Antichità Romane, scrive non poter affermare se
Valerio sia stato il primo a pronunciare in Roma un discorso funebre,
o s’egli abbia invece seguito un costume già invalso tra i re; ma in
ogni modo ritiene il costume come romano e rimprovera i tragici
ateniesi per averne voluto fare un merito alla loro città, che non
conobbe, a suo avviso, le orazioni funebri, se non dopo la battaglia
di Maratona, che fu posteriore di sedici anni alla morte di Bruto.
Ripigliava quindi la processione il suo corso e uscendo dalla
città [247] pel Circo Massimo e la porta Capena per mettersi nella via
Appia, ch’era quella delle tombe, si avviava al rogo, rogus, ed anche
grecamente pira, πυρά, che era una specie di altare, o catasta,
piuttosto costruita nel recinto sepolcrale detto bustum, o contiguo
alla tomba, di ciocchi d’alberi resinosi, non digrossati ne’ squadrati,
in masse ad angoli retti, adorna di ghirlande e ramoscelli di cipresso,
sulla cima della quale collocavasi dallo schiavo, detto Ustor, il
cadavere, asperso di preziosi liquori e avvolto in un lenzuolo di
amianto. Dapprima il più prossimo congiunto o l’erede ne aveva
riaperto gli occhi, come voleva il rito, reputandosi sacrilegio il privare
il cielo degli sguardi di un morto, e curavasi che portasse al dito il
suo anello e prima di avvilupparlo nel sudario la moglie e i figli
deponevano sulle gelide labbra di esso l’estremo bacio, tributo
ultimo che pur a sè stesso augurava ricevere dalla sua Cinzia
Properzio:

Osculaque in gelidis pones suprema labellis

Quum dabitur Syrio munere plenus onyx [248].

E si staccavano dalla cara spoglia colle parole consacrate dal rito:

Vale. Nos te ordine quo natura voluerit cuncti sequemur [249].
Ciò fatto, il più prossimo congiunto, stornando la testa, appiccava
colla rovescia torcia il fuoco alla pira e mentre questa ardeva,
gittavansi su d’essa incensi, profumi, vino, capelli e fiori, e lo stesso
Poeta or ricordato, pone in bocca alla sua Cinzia, che morte gli
toglieva anzi tempo e l’ombra della quale gli era ne’ sogni apparsa, il
lamento perchè nè di profumi, nè di vino e neppure di giacinti avesse
egli onorato il suo rogo:

Cur ventos non ipse rogis, ingrate, petisti?

Cur nardo flammæ non oluere meæ?
Hoc etiam grave erat nulla mercede hyacinthos
Inficere, et fracto busta piare cado [250].
Altri buttavan su quella fiamma le armi, le phaleræ [251] e le
vestimenta preziose del defunto, i cavalli a lui prediletti, i molossi, i
papagalli e quanto in vita aveva di meglio amato, e accadde ancora
che in mezzo ad essi si slanciassero gli schiavi stessi, come per
essere compagni al trapassato nel viaggio d’oltre tomba. Così
Virgilio menzionò nell’Eneide il devoto costume:

Hic alii spolia occisis direpta Latinis

Conjiciunt igni, galeas, ensesque decoros
Frænaque, ferventesques rotas: pars munera nota,
Ipsorum clypeos, et non felicia tela [253].

perocchè munera appunto si chiamassero le preziose cose avute in

pregio del suo vivente dal defunto.
Nè il popolo restava inoperoso in mezzo alla cerimonia; ma pregava
i venti spirassero secondi, giusta il costume de’ Greci rammentato
nell’Iliade nei funerali di Patroclo:

Ma del morto Patròclo il rogo ancora

Non avvampa, allor prende altro consiglio
Il divo Achille. Trattosi in disparte,
Ai due venti Ponente e Tramontana
Supplicando, solenni ostie promette,
E in aurea coppa ad amendue libando,
Di venirne li prega, e intorno al morto
Sì le fiamme animar, che in un momento
Lo si struggano tutto esso e la pira [254].

Se il funerale era di condottiero di esercito, cavalieri e fanti

riccamente ornati tre volte giravano intorno al rogo, ciò che
chiamavasi decursio, mandando dolorosi lai:

Ter circum accensos, cincti fulgentibus armis,

Decurrere rogos: ter moestum funeris ignem
Lustravere in equis, ululatusque ore dedere
Spargitur et tellus lacrymis, sparguntur et arma [255].
Così leggesi anche in Svetonio nella vita di Claudio, come intorno al
tumulo di Druso Germanico corresse ogni anno un soldato; e Tacito
scrive: Chatti tumulum super varianis legionibus structuri, ad aram
Druso sitam disiecerant. Restituit Cæsar arum honorique patris
princeps ipse cum legionibus decucurrit [256].
Ed intanto che il fuoco divampava, si facevano le libazioni di vino, di
latte e di sangue, alla quale ultima fornivanlo le vittime immolate, i
prigionieri, gli schiavi od anche i gladiatori detti bustuari, per ciò che
innanzi al rogo combattessero combattimento mortale, avendosi
fede, come già notai, che i mani si placassero col sangue.
I più ricchi e prestanti cittadini, siccome m’accadde di mentovare nel
capitolo dell’Anfiteatro nel dire de’ ludi gladiatorii, crescevano
onoranza con gli spettacoli di gladiatori, che si offerivano
gratuitamente al pubblico, e il dittatore Lucio Cornelio Silla li dispose
grandiosissimi pe’ suoi funerali nel proprio testamento, secondo si
raccoglie nell’orazione di Cicerone, pur da me già citata nei capitoli
della Storia Pompejana, recitata pro Publio Sylla.
Ma ad onorare la memoria dei defunti altri modi vi erano. La lettera
XVIII del Lib. VII. delle Epistole di Plinio il Giovane ci apprende come
Caninio Rufo, per eternare la memoria della propria moglie,
disponesse un annuale convito a’ suoi concittadini Comaschi e in un
rottame di lapide conservatoci dal Giovio si legge che altri due
Caninii della stessa città lasciassero similmente una somma per
celebrare un annuale banchetto: eccone il frammento:
ORNAMENTVM ET ROSA PONERETVR
RELIQ. INTER SE SPORTVLAS DIVIDERENT
IN CVIVS TVTEL. DEDERVNT CANINIVS VIATOR
ET CANINIVS EVPREPES HS.

Più munificente e assennato appare dalla medesima lettera XVIII

Plinio stesso, quando ci dice aver assegnato 500 mila sesterzi alla
sua patria Como per educare i giovani.
Le persone povere, o quelle non così facoltose e in grado di
comperarsi il bustum, portavansi all’ustrina, terreno publico destinato
a bruciare i cadaveri, le ceneri de’ quali venivano quindi trasportate
ne’ sepolcreti di famiglia se l’avevano, o se non l’avevano, al
sepolcro comune, al quale accenna Orazio nel verso:

Hoc miseræ plebi stabat comune sepulcrum [257];

perocchè la legge proibisse di appiccare il fuoco ad un rogo sul

terreno d’altrui proprietà.
Consumatasi la salma, estinguevansi le fiamme col vino: il più
prossimo parente, cantandosi da’ musici l’epicedum o poemetto
funebre in onore del morto, (da ἐπὶ sopra, e κὴδος, funerale),
raccoglieva le ossa ancora ardenti, le lavava in vecchio vino e nel
latte e le asciugava con un lino, ciò che chiamavasi ossilegium.
Siffatta costumanza di lavare nel vino, nel latte e talvolta anche
nell’olio le ossa, era stata vietata come inutile scialaquo dalle leggi
delle XII Tavole; ma non per questo era stata meno e sempre in
vigore e in Roma e presso tutte le nazioni a’ Romani soggette. Giova
anzi a tal proposito ricordare il grazioso epitaffio che uno schiavo
aveva scolpito sulla tomba da lui fatta erigere al giovinetto padrone
suo, che così si chiudeva:

Ossibus infundam quot numquam vina bibisti [258].

alludendo al divieto de’ Romani che i fanciulli avessero a bever vino.

Riponevansi da ultimo le ossa in un’urna talvolta di bronzo, il più
spesso di terra cotta, di marmo, di alabastro o di vetro, del quale
ultimo materiale è l’urna cineraria, scoperta in Pompei, riempita per
metà di un liquido e nel quale si discernono ancora i resti di ossa e di
ceneri. Oltre di tale liquido si sa vi ponessero rose e piante
aromatiche. Un sacerdote per ultimo aspergeva d’acqua lustrale i
parenti onde purificarli, ciò che dicevasi suffitio, e dopo, il capo della
cerimonia, designator, od anche la prefica, diceva loro: I licet, cioè
potete ardarvene e la comitiva si discioglieva. Allora chiudevasi
l’urna nella tomba, sulla quale ponevasi la pietra detta monimentum,
onde fu poi generalizzato il nome di monumento agli edifizi funebri e
sovr’esso l’inscrizione predisposta. Più avanti ne recherò parecchi
saggi di quelle trovate e lette negli scavi della Via delle Tombe in
Pompei.
E i vasi lacrimatorj, mi si chiederà, a che non li avete voi accennati,
prima di chiudere col monimentum il sepolcro?
È di fatto che ne’ sepolcri antichi, e pur in quelli di Pompei si
rinvenissero vasi e cucchiai detti lagrimatorii; ma servivano essi
davvero a raccogliere, come fu preteso, le lagrime de’ veraci dolenti
e delle prezzolate prefiche?
Il Baruffaldi lo credette nella sua Dissertazione De Præficis e lo
credettero il Fabbretti nel suo libro delle Iscrizioni ed altri ancora; e il
Fabbretti volle anzi da certi fori praticati sovente sul coperchio delle
antiche tombe, argomentare l’usanza d’introdurvi per essi le lagrime
de’ congiunti ne’ giorni anniversarii o nelle feste commemorative de’
loro cari defunti, molto più che in taluni vasi si sieno vedute delineate
le orbite degli occhi, e sui monimenta si riscontrino scolpite tazze ed
espresse le lagrime negli epitaffi; ma colla dovuta reverenza a questi
dotti, io non mi sono mai capacitato che tal costume avesse potuto
un giorno sussistere. Piagnone prezzolate, artifici di dolore per
quanto sottili, lagrime di dolenti, versate dopo parecchi giorni dal
decesso del caro parente, come avrebbero potuto fornir tanta
materia a’ cucchiai e vasi lacrimatorii? Questi arnesi, queste fiale di
vetro o di terra cotta, di alabastro o d’altro, non sarebbero stati
piuttosto adoperati a raccogliere balsami ed aromi che gittavansi sul
rogo, o libazioni di latte e di vino, che si facevano sulle tombe?
Il Grutero, nella eruditissima sua opera, recò più d’una iscrizione, fra
le cui parole vedevansi scolpite cucchiaj o patere, come più
propriamente dicevansi, le quali erano appunto vasi circolari con
manichi, atti a contenere liquidi, ma più specialmente usati, dice
Rich, a contenere il vino con cui era fatta una libazione [259].
I fori adunque praticati ne’ coperchi delle tombe debbono
indubbiamente aver servito a far penetrare le libazioni di vino e di
latte, di che Foscolo pur tenea conto in que’ versi de’ suoi Sepolcri:
Le fontane versando acque lustrali,
Amaranti educavano e viole
Su la funebre zolla; e chi sedea
A libar latte e a raccontar sue pene
Ai cari estinti, una fragranza intorno
Sentia qual d’aure de’ beati Elisi [260].

Questi fori si praticavano ad esempio nei loculi, o bare, quando i

cadaveri non si abbruciavano ancora ma si collocavano in essa
interi. Entro codesta bara spesso di terra cotta, eravi ad una
estremità una soglia elevata per adagiarvi il capo e dallato un foro
tondo pei balsami aromatici, che si versavano dentro per mezzo d’un
corrispondente orificio nella parete esterna della cassa.
Il costume della cremazione de’ cadaveri, del cui procedimento
presso i Romani ho intrattenuto il lettore, non era antico a’ tempi di
Plinio, voglio dire al tempo della catastrofe di Pompei, siccome egli
l’attesta. Era la cremazione usata solo in Grecia fin dai tempi di
Cecrope, dicendo Luciano che il Greco abbrucia i cadaveri, il
Persiano li sotterra, l’indiano li avvolge di grasso porcino, lo Scita li
divora, l’Egizio li imbalsama. L’essersi adottata da Romani originò
dall’oltraggio che veniva fatto alle tombe, quando i romani morivano
in lontane contrade, e che però vi si voleva ovviare. Lucio Cornelio
Silla, che a cagione delle tante proscrizioni aveva ragione a temere
l’insulto al proprio cadavere, com’egli stesso aveva fatto a quello di
Mario, dissotterrandolo e facendolo gittare nel Teverone, fu il primo
che ordinasse di ardere, dopo morte, il proprio corpo, e così invalse
il costume passato in legge, e Ovidio lo rammenta nel verso:

Corpora debentur mæstis exanguia bustis [261].

Siccome poi essi pensassero che l’anima fosse della natura del
fuoco, e che il rogo le facilitasse l’uscita dal corpo e però l’onore del
rogo non s’avesse a concedere che alle persone dotate di ragione e
sentimento; così, per testimonianza dello stesso Plinio il Vecchio,
non s’accordava a’ bambini, a’ quali non fossero ancora spuntati i
denti, perocchè sarebbe stata considerata siccome empietà che