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Histology 1

This document discusses the preparation of histological specimens. It describes the various stages of processing tissues, including fixation, dehydration using ethanol or other agents, clearing with xylene or other substances, and embedding in paraffin wax. Sections are then cut from the embedded tissue using a microtome and stained, often with hematoxylin and eosin, for examination under a microscope. Histology is the study of tissues and how they are arranged into organs at a microscopic level. Proper preparation of specimens is necessary to reflect the actual nature of tissues and enable examination.

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0% found this document useful (0 votes)
160 views5 pages

Histology 1

This document discusses the preparation of histological specimens. It describes the various stages of processing tissues, including fixation, dehydration using ethanol or other agents, clearing with xylene or other substances, and embedding in paraffin wax. Sections are then cut from the embedded tissue using a microtome and stained, often with hematoxylin and eosin, for examination under a microscope. Histology is the study of tissues and how they are arranged into organs at a microscopic level. Proper preparation of specimens is necessary to reflect the actual nature of tissues and enable examination.

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raphael
Copyright
© © All Rights Reserved
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PREPARATION OF HISTOLOGICAL SPECIMENS rigidity to enable thin sections to be cut, and yet soft

enough not to damage the knife or tissue.


 Histology is the study of tissues and how these
tissues are arranged into organs Stages of processing:
 Histo in Greek means tissue or web
 Dehydration.
 Tissues consist of cells and extra cellular matrix
 Clearing.
 The function of the tissue depends on the
 Embedding.
interaction between cells and extracellular
matrix. Dehydration:
 The small size of cells and matrix content make
the study of tissues dependent on microscope  To remove fixative and water from the tissue
and other advances in biological techniques and replace them with dehydrating fluid.
 Delicate specimens are dehydrated in a graded
TISSUE PREPARATION ethanol series from water through 10%-20%-
50%-95%-100% ethanol to minimize tissue
 The most widely used method of studying
distortion from diffusion currents.
tissues is using histological slides.
 In the paraffin method, dehydration from
 The tissue in the slide must reflect the actual
aqueous fixatives is usually initiated in 60%-
nature of the tissue in the body
70% ethanol, progressing through 90%-95%
 To insure that, tissues to be studied must pass
ethanol, absolute ethanol before proceeding to
through a series of steps before examination
the clearing stage.
 These steps are in sequential order
 Fixation is a complex series of chemical events Types of dehydrating agents
that differ for the different groups of substance
found in tissues.  Ethanol
 Methanol
TISSUE FIXATION  Acetone
Aim of Fixation: Tissues may be held and stored indefinitely in 70%
ethanol without harm
1- To prevent autolysis and bacterial attack.
2- To fix the tissues so they will not change their Clearing
volume and shape during processing.
3- To prepare tissue and leave it in a condition  Replacing the dehydrating fluid with a fluid that
which allow clear staining of sections. is totally miscible with both the dehydrating
4- To leave tissue as close as their living state as fluid and the embedding medium.
possible, and no small molecules should be lost.  Choice of a clearing agent depends upon many
factors
Fixation is a reaction between the fixative and proteins
in the specimen which form a gel, so keeping everything Types of Clearing Agents:
as their in vivo relation to each
 Xylene.
Types of Fixative:  Toluene.
 Chloroform.
 Acetic acid  Benzene.
 Formaldehyde 10%  Propylene oxide
 Ethanol
 Glutaraldehyde Embedding
 Methanol
 Is the process by which tissues are surrounded
 Picric acid
by a medium such as agar, gelatin, or wax which
 Osmic acid (Osmium tetroxide) when solidified will provide sufficient external
TISSUE PROCESSING support during sectioning.
 A substances added alone or in combination to
Aim: Is to embed the tissue in a solid medium firm the wax to:
enough to support the tissue and give it sufficient o Improve ribboning.
o Increase hardness.
o Decrease melting point Staining machine
o Improve adhesion between specimen
and wax Basic dyes stain:

Embedding Moulds:  Heterochromatin


 Nucleic acids
 paper boat mould  Ribosomes
 metal boat mould  Cartilage
 Dimmock embedding mould
 Peel-a-way disposable mould Acidic dyes stain:
 Base mould used with embedding ring ( F) or  Filaments
cassette bases (G)  Mitochondria
CUTTING - using the microtome  Collagen
 Muscle fibers
 A microtome is a mechanical instrument used
to cut biological specimens into very thin Additional Dyes
sections for microscopic examination.  Many tissue components can not be stained
 Most microtomes use a steel blade and are with (Hematoxylene and Eosin).
used to prepare sections of animal or plant  Other dyes are used to specifically stain certain
tissues for histology. tissue components Resorcin-Fuchsin for elastic
Microtome knives fibers Silver stain for reticular fibers and
basement membrane Periodic-Acid Schiff (PAS)
 Steel knives Reaction for CHO
 Non-corrosive knives for cryostats
 Disposable metal blades
 Glass knives
 Diamond knives
STAINING
Hematoxylin and Eosin (H & E)

 H & E is a charge-based, general purpose stain.


 Hematoxylin stains acidic molecules shades of
blue.
 Eosin stains basic materials shades of red, pink
and orange.
 H & E stains are universally used for routine
histological examination of tissue sections.
Staining Procedure:

 Deparaffinize and hydrate to water


 If sections are Zenker-fixed, remove the
mercuric chloride crystals with iodine and clear
with sodium thiosulphate (hypo)
 Mayer's hematoxylin for 15 minutes
 Counterstain with eosin from 15 seconds to 2
minutes depending on the age of the eosin, and
the depth of the counterstain desired
 Dehydrate in 95% and absolute alcohols, two
changes of 2 minutes each or until excess eosin
is removed
 Clear in xylene, two changes of 2 minutes each
 Mount in Permount or Histoclad
“HISTOLOGY” - the science of the tissues Purkinje (1787-1869), a Czech biologist, in
1830.
 histos is Greek for web or tissue
 Robert Brown (1773-1858), a Scottish botanist
 logia is Greek for branch of learning
introduced the term nucleus in microscopy
 Tissue was first used to describe the different after the examination of epidermal cell of some
textures of body parts being dissected by an orchids and some Asclepiadacea in 1831.
anatomist.
Cell as the fundamental unit that constitutes all the
“Microanatomy” generality of animals and plants
 the study of the structure (ANATOMY) of small  Matthias Schleiden (1804-1881), a German
(MICRO) things. botanist, in 1838; He saw, under the
 small things cells and their arrangement to microscope, thousands of plant specimens, and
constitute tissues and the association among inferred that all the vegetables are made of
these to form organs. cell.
HISTOLOGY  Theodor Schwann (1810-1882), a German
History and Background zoologist and physiologist, in 1839; He came to
the conclusion that all the then known living
Who discovered the cell? beings were composed of cells. “CELL THEORY”
Robert Hooke (1635-1703) All living cells originated from pre-existing ones.
 Micrographia, published in 1665 Rudolf Virchow (1821-1902),
 upon examining a piece of cork with a
rudimentary microscope, saw an abundance of  a great German pathologist demonstrated that
empty small compartments the pathological injuries also had a cellular
 the “cell” was discovered! structure.
 cell walls in cork tissue – empty cell  the pioneer of the cellular pathology.
 Latin, cellula;“ CELL”  edified the bases of the modern Pathology.
 In the sequence of the works of Theodor
 History and Background Schwann, Virchow saw (1855/56) that all the
cells of the pathological tissues are derived
Transition from "empty cell” to the actual cell
from healthy ones and that all living cells
 1665 – same year as discovery of cell, Hooke originated from pre-existing ones.
and Marcello Malpighi were the first to  Law of Virchow (or Fundamental Law of
observe the true units that form the tissues of Biology)
animals.  Omnis cellula e cellula ; that is, each cell comes
 1678, after Leeuwenhoek reported of from another cell (1858).
discovering "little animals” (animalcules) -- “TISSUE” - - texture
bacteria and protozoa to the Royal Society.
 Hooke confirmed Leeuwenhoek’s findings.  “21 Textures” of Marie François Bichat
 Hooke noted that Leeuwenhoek's simple  a French pathologist (1771-1802)
microscopes gave clearer images than his  Bichat verified that certain textures presented a
compound microscope, but found simple thin thickness and were very flat, to the extent
microscopes difficult to use. of being compared to pieces of cloth.
 (French, tissu - means to weave).
Discovery of nucleus
 The first notion of “tissue”.
 The first description of the nucleus was carried  A texture was a “tissue” (a body component) as
out by Leeuwenhoek, in 1700, when examining perceived by its macroscopic physical
the red blood cells of the salmon. properties.
 The first description of the nuclear envelope
was accomplished by Jan Evangelista
The Father of Histology
Bichat vs Malpighi

 Bichat is considered, by some authors, to be


The first textbook of Histology
the founder of Animal Histology. He is virtually,
the first histologist.  Handbuch der Gewebelehre des
BUT…. Menschen
 (The book for teaching tissues); a landmark
 Marcello Malpighi (1628-1694), an Italian textbook
anatomist considered the true “Father of  Author: Rudolph Albert von Kölliker(1817-
Histology”. 1905) a Swiss professor of Anatomy published
in 1852
WHY???
Leeuwenhoek could not be the inventor of the
 Malpighi preceded Bichat in a number of years;
microscope, nor Malpighi.
 Bichat never used a microscope; His textures
were described based on dissections.  Hans Janssen (died about 1595) & Zacharias
 Although Malpighi used a rudimentary Janssen (1580? -1640) - The first who
microscope, he described a series of designed a microscope in the Netherlands late
microscopic structures. in the 16th century were working as spectacle
 He was the first scientist to observe the makers.
capillaries.  Hans and his son Zacharias Janssen are
Marcello Malpighi mentioned in the letters of William Boreel
(the Dutch envoy to the Court of France) as
 also called the "Father of Microscopic having invented a 20X magnification
Anatomy" microscope.
 Malpighi performed his greatest feat when  However Jan Swammerdam, Robert Hooke
he observed blood circulating through and Malpighi were the first scientists who
capillaries and recorded what he saw. used microscopes for their research in
Definition of “tissue” histology.

 Tissue is each of the elementary multicellular Modern: Compound Light MICROSCOPE


components, microscopically and functionally Calibration factors:
distinct, that constitute either animals or plants
where they associate to form organs and  Scanner = 125m
systems.  LPO = 50 m
 HPO = 12.5 m
“HISTOLOGY”  OIO = 5 m
 August Mayer (1787-1865) coined the term  Size of object = no. ocular units X CFused
histologie Histological Anatomy
 from the Greek words (histos, "web" or "warp")
and (logos, "word" or "treatise")  Advances in chemistry, physiology,
 to mean "the study of the tissues of the body” immunology, and pathology—and the
 The term came into common usage in the interactions among these fields—are essential
1840s. for a better knowledge of tissue biology.
 In 1844, it was recommended for large usage  Familiarity with the tools and methods is
by Sir Richard Owen (1804-1892), a great essential for a proper understanding of the
English palaeontologist. subject.
 The English word histology probably came from How tissues are prepared for observation
the German histologie via the French histologie.
 The preparation of tissues for microscopic
examination
 For light to pass through, tissues and organs up with a slide coated with a thin film of albumin or
must be sectioned to obtain thin, translucent Haupt’s adhesive and allowed to dry.
sections. 7. Staining – use of dyes to penetrate into
 [However, living cells, very thin layers of tissues, translucent tissue section. Basic and acidic dyes
or transparent membranes of living animals (Ex: impart contrast to micro-sectioned tissues allowing
mesentery, tadpole tail, the wall of cheek) can the observer to study the cells and its ultra-
be observed directly in the microscope without structures and the matrix.
micro-sectioning.]
 Most tissues need thin slices using microtome. Behavior of tissues to dyes
 This method preserves tissues in longer  Hematoxylin and Eosin (H&E) – commonly
duration under varying physiological or used dyes
experimental conditions.
 Basic dyes – hematoxylin, toluidine blue,
Microtome methylene blue stain basophilic structures such
as nucleus, free ribosomes, matrix of hyaline
Histotechniques for paraffin sections using rotary cartilage.
microtome  Acid dyes – orange G, eosin, acid fuchsin stain
 Fixation – prevents autolysis (self-destruction) the acidophilic components of tissues such as
or bacterial invasion of tissues leading to tissue mitochondria, secretory granules, cytoplasm
distortion and degradation and collagen.
 Use of fixative chemicals as stabilizers or cross-  Trichromes – differentiating stains
linkers maintaining the molecular and structural  Counterstain – a single stain that is applied to
composition of tissues. a section to allow the recognition of nuclei or
 Formaldehyde and glutaraldehyde are cytoplasm, is used.
widely used fixatives which react with the  Metallic dyes – silver or gold is ideal for
amine groups (NH2) of tissue proteins. nervous tissue.
Glutaraldehyde, fixing action is reinforced since
it is a dialdehyde, can cross-link proteins.
Tissue processing–Involves dehydration (ethanol) and
clearing (xylene).

1. Dehydration - It is the first part of the process. It is


usually accomplished by transferring the block of
tissue through a series of alcohol-water solutions
beginning with 50 percent and running up to
water-free or absolute alcohol.
2. Clearing - The alcohol is replaced by xylene or
cedar oil, which is readily soluble in alcohol, and in
turn, is replaced by melted paraffin.
3. Paraffin Impregnation – melted paraffin
infiltrates the tissue gradually.
4. Embedding - paraffin- infiltrated tissue is placed in
fresh paraffin and the latter allowed to cool.
 Paraffin or plastic resins give a rigid
consistency to the tissue.
 Result: Tissue block - solid to facilitate
sectioning.
5. Tissue sectioning – mounting the tissue block in
the microtome
6. Affixing section on slide – section is floated on
a warm water-bath to expand / flatten; and picked

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