The document discusses the process of preparing tissue samples for microscopic analysis. It explains that cells are organized into tissues, organs, and organ systems within multicellular organisms. The preparation process involves fixing, dehydrating, clearing, embedding, sectioning, and staining tissue samples. This allows histologists to examine tissues at the microscopic level and study their structure and composition. Key steps include fixing tissues with chemicals to preserve their structure, embedding samples in paraffin wax for sectioning, and staining slices with dyes to differentiate between components for examination under a microscope.
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INTRODUCTION TO VETERINARY HISTOLOGY I
1.
2. Overview of body organization
Cells are considered the fundamental units of life.
All living organisms are made up of one or more cells.
Unicellular organisms, like amoebas, consist of only a single
cell.
Multicellular organisms (Metazoa), like animals, are made
up of many cells.
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3. The cells in metazoan organisms are organized into tissues:
groups of similar cells that work together on a specific task.
Organs are structures made up of two or more tissues
organized to carry out a particular function, and
organ systems are a groups of organs with related functions.
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4. Histology is a branch of anatomy that deals with the study of
tissues and cells that can be seen only with the aid of a
microscope.
The term is derived from the Greek "histos" meaning web or
tissue, and refers to the "science of tissues".
It is also known as microscopic anatomy.
Histopathology, the study of tissues affected by disease, can
be very useful in making a diagnosis and in determining the
severity and progress of a condition.
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5. Obviously, if an organ were to be examined, it would be
impractical to place the entire organ under a routine light
microscope for study.
It is not only much too large, but also opaque, therefore an
examination of its micro-components would be impossible.
For this reason and several others, a small portion of a specific
tissue or organ must be excised from a given organ and
processed for microscopic analysis.
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6. Various techniques have been developed to prepare tissues for
study so that they closely resemble their natural, living state.
The ideal microscope tissue preparation should be preserved
so that
the tissue on the slide has the same structure and molecular composition
as it had in the body.
this is sometimes possible but – as a practical matter – seldom feasible,
and artifacts, distortions, and loss of components due to the preparation
process are almost always present.
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7. In order to study tissues with a microscope they must be
preserved (fixed) and cut into sections thin enough to be
translucent.
Techniques needed to preserve the structural integrity of a
specimen so that it can be viewed microscopically.
The process through which cell structure is preserved is called
fixation.
Fundamentally it consists of a chemical or physical method of killing
the tissue and yet retaining characteristic peculiarities of shape and
structure.
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8. 2.1. Procedures in Histotechniques
aim to provide good quality sections that can be used for a
light microscopic evaluation animal tissue.
Tissue processing describes the steps required to take
animal tissue from fixation to the state where it is completely
infiltrated with a suitable histological wax and can be
embedded ready for section cutting on the microtome.
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9. Routinely,
tissues are fixed with neutral formalin 10%,
Processed,
embedded in paraffin, and
then manually sectioned with a microtome to obtain 4–5
micrometer thick paraffin sections.
Dewaxed sections are then stained with hematoxylin and eosin
(H&E) or can be used for other purposes (special stains,
immunohistochemistry, in situ hybridization, etc.).
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11. 1) Fixation
Fixation is the first step in any procedure in which tissue is to
be preserved for histological study.
It refers to treatment of tissue with chemical agents/ physical
that not only retard alterations of tissue subsequent to death (or
after removal from the body) but also maintain its normal
structure.
Fixatives:
kill the tissue, as well as any bacteria that are present that otherwise
would cause the tissue to rot.
Coagulate or cross-link proteins, making them insoluble, halt autolysis
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12. rapid and adequate fixation after sampling is essential as the
process of autolysis set of immediately after death .
the most common fixative agents is 10% neutral buffered
formalin (4% aqueous solution of formaldehyde).
___Others, mercurials, alcohols, oxidizing agents, and picric
acid derivatives.
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13. 2) Dehydration and clearing
the collected tissues, once fixed, are then dehydrated in graded
solutions of alcohol (ethanol is preferable to methanol, since it
is less harsh on the tissues ) or other dehydrating agents,
a graded series of alcohol baths, beginning with 50% alcohol
and progressing in graded steps to 100% alcohol, are used to
remove water (dehydration), w/c is immiscible with paraffin.
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50% 60% 70% 80% 90%
14. Once tissue is dehydrated, a clearing agent, such as xylene or
toluene, which is miscible with both 100% alcohol and
paraffin, makes a bridge between the alcohol and paraffin.
xylene is routinely used for clearing tissues.
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100%
alcohol
xylene
15. 3) Embedding
Commonly used embedding medium is paraffin.
paraffin embedding replaces tissue water with paraffin wax,
enabling the block to be cut readily.
the xylene-permeated block is passed through several changes
of warm paraffin wax, which is soluble in xylene.
after the tissue is impregnated with paraffin, it is placed into a
small receptacle/tissue casette, covered with melted paraffin,
and allows to harden, forming a paraffin block containing
tissue.
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16. 4) sectioning
after the formed paraffin blocks together with the contained
tissues are trimmed of excess embedding material, they are
mounted for sectioning on a cutting device called a
microtome.
Mounting sections- the cutting process compresses the
sections, and part of the mounting procedure is to expand the
sections before they adhere to the slide.
Is done floating the sections on warm water (5–10ºC below the melting
point of the paraffin).
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17. Mounting the ribbons- Ribbons of serial sections must be
placed in order, one under the other, until the width of the slide
is filled.
5) Staining
Because many tissue constituents have approximately the
same optical densities, they must be stained for light
microscopy:
aqueous solutions are usually used in staining. Prior to
this, the wax must be removed (dissolved) and replaced
with water.
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18. This is accomplished by passing the slides together with their
mounted sections through xylene or toluene
to remove paraffin and then through descending strengths of
alcohol solutions to water, as most of the dyes used are in
aqueous solutions.
although various types of stains have been developed
for visualization of many components of cells and
tissues, they may be grouped into three classes:
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19. 1) Stains that differentiate between acidic and basic
components of the cell,
2) specialized stains that differentiate fibrous components of the
extracellular matrix,
3) metallic salts that precipitate on tissues forming metal
deposits on them.
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20. the most commonly used stains in histology.
Hematoxylin is a base that preferentially colours the acidic
components of the cell a bluish tint,
because the most acidic components are deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA),
the nucleus and regions of the cytoplasm rich in ribosomes stain dark
blue, these components are referred to as basophilic.
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21. Eosin is an acid that dyes the basic components of the cell a
pinkish color,
because many cytoplasmic constituents have a basic pH,
regions of the cytoplasm stain pink; these elements are said to be
acidophilic.
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22. Observation of tissues under a light microscope is an old
concern for science and medicine.
The earliest evidence of magnifying glass forming a magnified
image dates back to 1021 when the physicist Ibn al-Haytham
(965–1039) published the “Book of Optics.”
The name “microscope” was crafted by the German botanist
Johann Faber (1574–1629).
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23. The light microscope used by Anton Van Leeuwenhoek’s
(1632–1723) was a small, single convex lens mounted on a
plate.
The light microscope is the tool used most widely for clinical
applications of histology.
the advent of the electron microscope greatly extended the detail
at which subcellular structure can be studied.
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