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LECTURE NOTES

Course Title: Histology and Cytology

Course Code: MLT 3001
Level: Year 3 HND students
Semester: 1
Master: By Mr. Tadoum Talla Christian

Course Aim: The Course is designed to give an understanding on the nature of cells
of the body, tissues and their functions, processing and examination of tissues and
cells in histopathology.

Expected Outcomes: At the end of this course students are supposed to:

 Histological and cytological staining techniques

 To study tissues sections under the microscope.

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Table of content

Table of Contents
CHAPTER ONE..............................................................................................................................3
Understanding the Cell....................................................................................................................3
1.0 Introduction to histology........................................................................................................3
Introduction..................................................................................................................................3
Cell Theory consists of three principles:.................................................................................3
CELL SIZE..............................................................................................................................4
CELL SHAPE..........................................................................................................................4
INTERNAL ORGANIZATION..................................................................................................4
Organelles and their functions.....................................................................................................5
A) Cell Membrane...................................................................................................................5
B) CYTOPLASM....................................................................................................................6
C) THE NUCLEUS.................................................................................................................6
D) MITOCHONDRIA.............................................................................................................7
E) RIBOSOMES......................................................................................................................8
F) ENDOPLASMIC RETICULUM (ER)...............................................................................8
G) GOLGI APPARATUS.......................................................................................................9
H) LYSOSOMES....................................................................................................................9
I)CYTOSKELETON.............................................................................................................10
J) CENTRIOLE.....................................................................................................................10
K) CILIA AND FLAGELLAE..............................................................................................10
CHAPTER TWO...........................................................................................................................12
TISSUES........................................................................................................................................12
2.0 Introduction..........................................................................................................................12
2.1 EPITHELIUM.....................................................................................................................12
INTRODUCTION TO THE CONCEPT OF EPITHELIUM................................................12
2.2 FEATURES OF EPITHELIUM..........................................................................................12
2.3 FUNCTIONS OF EPITHELIUM........................................................................................13
2.4 MORPHOLOGICAL CLASSIFICATION OF EPITHELIA..............................................14
1) SIMPLE EPITHELIA.......................................................................................................14
2) STRATIFIED EPITHELIUM...........................................................................................16
GLANDULAR EPITHELIUM.............................................................................................19
CHAPTER THREE.......................................................................................................................21
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CELL JUNCTIONS/CONNECTIVE TISSUES...........................................................................21

Introduction............................................................................................................................21
3.2 CONNECTIVE TISSUES...................................................................................................22
3.2.1 Functions of connective tissues....................................................................................22
3.2.2 Classification of Connective tissues.............................................................................22
3.3 Understanding the architectural make-up of loose connective tissues:...............................22
3.4 Dense Connective tissues....................................................................................................25
2- Supporting Connective Tissue...........................................................................................27
Types of supporting tissues...................................................................................................27
Types of cartilage:.................................................................................................................27
Bone / Osseous Tissue...........................................................................................................28
Composition...............................................................................................................................28
3.5 BONE CELLS.....................................................................................................................29
CHAPTER FOUR.........................................................................................................................31
EXAMINATION OF TISSUE IN A HISTOPATHOLOGY LABORATORY............................31
4.0 Introduction..........................................................................................................................31
4.1 Examination of fresh specimens..........................................................................................31
CHAPTER FIVE...........................................................................................................................32
Tissue Processing...........................................................................................................................32
5.1 Fixation of tissues................................................................................................................32
5.2 Actions of Fixatives.............................................................................................................33
A. Methods of Stabilizing Proteins........................................................................................33
B. Coagulant and Non-Coagulant..........................................................................................33
5.3 Some factors that affect fixatives........................................................................................34
5.4 Simple Fixatives used in the histopathology laboratory......................................................34
a) FORMALDEHYDE..........................................................................................................34
b) OSMIUM TETROXIDE...................................................................................................35
c) ACETONE.........................................................................................................................35
5.5 Compound Fixatives used in the histopathology laboratory...............................................35
A) ACETIC ACID.................................................................................................................35
B) PICRIC ACID..................................................................................................................35
5.6 MICROTOME.....................................................................................................................35
5.6.1Types of microtomes.....................................................................................................36
C. Cryomicrotome.................................................................................................................36

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5.6.2 Types of Microtome Blades..........................................................................................37

5.6.3 Technique of Cutting....................................................................................................38
5.6.4 Faults in paraffin section cutting..................................................................................38
CHAPTER SIX..............................................................................................................................40
STAINS USED IN A HISTOLOGY LABORATORY.................................................................40
6.0 Introduction..........................................................................................................................40
6.2 Terminologies in staining:...................................................................................................40
6.2Characteristics of Commonly used Stains............................................................................41
CHAPTER SEVEN.......................................................................................................................42
DECALCIFICATION...................................................................................................................42
7.0 Introduction..........................................................................................................................42
Decalcification protocol.........................................................................................................43
Monitoring End-Point of Decalcification:.............................................................................43
CHAPTER EIGHT........................................................................................................................45
BASIC TECHNIQUES.................................................................................................................45
Preparation of histological sections.......................................................................................45
8.0 Introduction..........................................................................................................................45
1. Fixation.............................................................................................................................45
2.0 Dehydration.....................................................................................................................45
3.0 Clearing............................................................................................................................46
4.0 Embedding/Impregnation................................................................................................46
4.1Frozen sections.................................................................................................................46
3. Microtomy........................................................................................................................47
4. Staining.............................................................................................................................47
4.1 H& E staining Technique................................................................................................47
5. Permanent Mounting........................................................................................................49
Types of mountants:..............................................................................................................50
SECTION II...................................................................................................................................51
Cytologic Techniques....................................................................................................................51
1.0 Introduction..........................................................................................................................51
Histology of the cervix..............................................................................................................51
Transformation Zone.................................................................................................................51
Collection of cytological specimens..........................................................................................52
1.2 Fixing of cytological specimens..........................................................................................52

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1.3 Staining cytological specimens............................................................................................52

1.3.1 Papanicolaou staining...................................................................................................53
1.4 Papanicolaou staining Technique....................................................................................53
3. Eosin Alcohol 50 (EA50).................................................................................................54

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CHAPTER ONE

Understanding the Cell

1.0 Introduction to histology

The name "Histology" is derived from the Greek word for a tissue "Histos", and "-logos" = the
study of. Today the concept of histology as a subject includes far more than just the study of
tissues, but includes understanding of the structure and function of cells, tissues, organs and
organ systems, which can better be described as "Microscopic Anatomy". In addition to
understanding the histology and ultrastructure of cells, tissues and organs, it is also necessary to
complement the morphological observations with an understanding of pathologies associated
with tissues and organs-histopathology

For students to understand histology, they should however understand the cell and it’s
importance in this course.

INTRODUCTION
Cells are the fundamental unit of living things--they are the smallest structures that show all the
features of living things. All organisms consist of small cells, typically too small to be seen by a
naked eye, but big enough for an optical microscope. Each cell is a complex system consisting
of many different building blocks enclosed in membrane bag. There are unicellular (consisting
only of one cell) and multicellular organisms. Bacteria and baker’s yeast are examples of
unicellular organisms - any one cell is able to survive and multiply independently in appropriate
environment.
There are estimated about 6x1013 cells in a human body, of about 320 different types. For
instance, there are several types of skin cells, muscle cells, brain cells (neurons), among many
others. The number of cell types is not well-defined, it depends on the similarity threshold
(what level of detail we would like to use to distinguish between the cell types, e.g., it is
unlikely that we would be able to find two identical cells in an organism if we count the number
of their molecules). The cell sizes may vary depending on the cell type and circumstances. For
instance, a human red blood cell is about 5µ (0.005 mm) in diameter, while some neurons are
about 1 m long (from spinal cord to leg). Typically the diameter of animal and plant cells are
between 10 and 100 microns.
Viruses are not quite living organisms, but when inside a living host cell they show some
features of a living organism. Viruses are too small to be seen in an optical microscope, but are
big enough to reveal their structure in an electron microscope (the characteristic size of the
virus is about 0.05-0.1µ, while the wavelength of green light is about 0.5µ).
The theories of cell origin
Before the invention of microscopes there was no way to observe the small-scale structure of
living things. Starting in the 17th century technology began to develop to enable scientists to
see the cells of microbes, and to see the cellular structure of larger animals. Robert Hooke

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(1665) coined the term "cell" while looking at the nonliving tissue known as cork.
In 1675, Anton van Leeuwenhoek, a Dutch lens maker, first reported observations of living
cells which he called "animicules". Robert Brown (1831) was the first to report the discovery
the nucleus of the cell. The understanding that larger animals were multicellular was relatively
slow in develop. It was not until the 19th century that the idea that all animals and plants were
constructed of cells, and that cells have a kind of life-cycle of their own was thought
of. Matthias Jakob Schleiden (1838), a German Botantist, after extensive studies reported that
all plants were composed of fundamental units known as cells. In 1839, Theodore Schwann, a
German Zoologist, after extensive studies reported that all animals were composed of
fundamental units known as cells. Rudolf Virchow (1858) proposed that all cells came from
pre-existing cells. The basic tenets of the cell theory:
 All organisms are composed of cells
 Cells are the basic unit of life
 Cells arise only from other pre-existing cell

Types of Cells
There are two types of organisms - eukaryotes and prokaryotes, and two types of cells
respectively. The term "eukaryote" means "true nucleus" while "prokaryote" means "before
the nucleus". This emphasizes the central importance of the nucleus to the eukaryotic cell, and
suggests that prokaryotes are more primitive organisms, and that eukaryotes evolved from them
by, among other things, acquiring a true nucleus. The both do have DNA for genetic material,
have a exterior membrane, have ribosomes, accomplish similar functions, and are very diverse.
For instance, there are over 200 types of cells in the human body, that very greatly in size,
shape, and function.
Prokaryotes Eukaryotes

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 Unicellular  Unicellular or multicellular

 Nucleus absent  Nucleus present
 Genetic material is not enclosed within a membrane  Genetic material is
 Do not engulf solids surrounded by a membrane
 Do not have centrioles or asters  Engulf solids
 Do not have internal membrane-bound organelles  Have centrioles or asters
 Smaller than eukaryotes (diam 1µ)  Do not have internal
 E.g. bacteria and cyanophyte membrane-bound organelles
 Larger than prokaryotes
 E.g. humans, algae, protazoa
Bacteria belong to the prokaryotes. However, most organisms which we can see, such as trees,
grass, flowers, weeds, worms, flies, mice, cats, dogs, humans, mushrooms and yeast are
eukaryotes. The distinction between eukaryotes and prokaryotes is rather important, because
many of the cellular building blocks and life processes are quite different in these two organism
types. This is believed to be the result of different evolutionary paths.
Prokaryotes are the simplest cells. Prokaryotic cells are smaller than eukaryotic cells (a typical
size of a prokaryotic cell is about 1 micron in diameter) and have simpler structure (e.g., they do
not have any inner cellular membranes that are always present in Eukaryotes). Prokaryotes are
single cellular organisms, but note that being a single cell does not mean that an organism is a
prokaryote. Being smaller than eukaryotes does not mean that prokaryotes are any less
important – for instance it is quite likely that the number of bacteria living in the mouth and
digestive tract of a human are larger than the number of eukaryotic cells in the same individual
and many of these bacteria are necessary for a human being to live a normal life (these numbers
are rather difficult to estimate, rather a hypothesis). Prokaryotes are sometimes also known as
microbes.
 Prokaryotic cells have the simplest overall structure.
 The prokaryotic cell is bounded by a lipid bilayer membrane, but does not contain any
internal membrane-bound organelles. It contains a region rich in DNA called
a nucleoid.
 The nucleoid contains a circular molecule of DNA which is the cells genetic material,
or genome.
 Surrounding the nucleoid is a region of cytoplasm rich in ribosomes, small protein-RNA
structures which do the job of synthesizing proteins. Finally, surrounding the plasma
membrane is a cell wall.
 Prokaryotes can have surface appendages which do particular jobs. Flagella are used for
locomotion. Pili are used for sexual reproduction (mating)
 One of the more surprising aspects of cell structure is the way that DNA molecules are
packaged into cells.
 The genome of a common bacterium like the gut bacterium Escherichia coli is about 1.5
millimeters in length, but the length of the bacterium is about 1000 times shorter.
 In the nucleoid, the DNA is both compacted so as to fit within the bacterium, and yet is
still accessible to the machinery necessary both to read the genetic instructions
(synthesize RNA) and to copy the DNA (replicate).
Eukaryotic cells are much more complex.
 By contrast to prokaryotic cells, eukaryotic cells are much more complicated. Much of
the cell is taken up by subcellular organelles bound by their own membranes (analogous

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to the plasma membrane surrounding the cell)

 Most importantly, eukaryotic cells have a nucleus which contains all of the cell's DNA.
In fact, .
 The problem of packaging DNA is greater in eukaryotes. A human genome would
stretch about 1 meter, but must fit in a cell 50,000 times smaller.
 However, eukaryotic cells (which included plants, animals, and fungi) include many
other subcellular structures with important roles in cellular metabolism
Cell content

Cells are 90% fluid (cytoplasm) which consists of free amino acids, proteins, glucose, and
numerous other molecules. The cell environment (ie. the contents of the cytoplasm, and the
nucleus, as well as, they way the DNA is packed) affect the gene expression/regulations, and
thus are very important parts of inheritance, below are approximations of other components:
Elements:
 59% Hydrogen (H)
 24% Oxygen (O)
 11% Carbon (C)
 4% Nitrogen (N)
 2% Others - Phosphorus (P), Sulphur (S), etc.
As far as molecules that make up the cell:
 50% protein
 15% nucleic acid
 15% carbohydrates
 10% lipids
 10% Other
What is inside the cell is the cytoplasm which is:
 Cytosol - a lot of water - all except the organelles.
Organelles (which also have membranes) in 'higher' eukaryote organisms:
 Nucleus (in eukaryotes) - where genetic material (DNA) is located, RNA is transcribed.
Nucleolus is rich in RNA and site of ribosomal RNA synthesis and assembly.
 Endoplasmic Reticulum (ER) - Membranous structure important for protein synthesis.
It is a transport network for molecules destined for specific modifications and locations.
There are two types:
o Rough ER - Has ribosomes, and tends to be more in 'sheets'. Synthesis of
exported proteins.
o Smooth ER - Does not have ribosomes and tends to be more of a tubular
network. Synthesis of lipids.
 Ribosomes - Half are on the Endoplasmic Reticulum, the other half are 'free' in the
cytosol, this is where the RNA goes for translation into proteins.
 Golgi Apparatus - Membranous structure important for glycosylation and secretion.
Intermediate in protein export.
 Lysosomes - Digestive sacks - the main point of digestion, these are only found in
animal cells.
 Peroxisomes - Use oxygen to carry out catabolic reactions, in both plant and animals.
 Microtubules - Made from tubulin, and make up centrioles,cilia,etc.

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 Cytoskeleton - Comprises microtubules, actin and intermediate

filaments; supports internal structures and gives shape to the cell.
 Mitochondria - convert foods into usable energy. (ATP
production) A mitochondrion does this through aerobic
respiration. They have 2 membranes, the inner membranes shapes
differ between different types of cells, but they form projections
called cristae. The mitochondrion is about the size of a bacteria,
and it carries its own genetic material and ribosomes.
 Vacuoles - Storage organ for storage of nutrients & water. More
commonly associated with plants which commonly have large
vacuoles.

Found in Plants and not in animals:

 Plastids - Specialised organelles found only in Algae and Plants; include chloroplasts,
chromoplasts and amyloplasts. Chloroplasts contains stacks of flattened membranes
specialized in photosynthesis. Chromoplasts store pigments which color plant parts
(e.g. petals). Plastids which store starch (e.g. in roots) are the amyloplasts, which are
also called leukoplasts.
 Cell Wall - found in prokaryotic plants and it provides structural support and protection

A model of an eukaryotic cell

 The most important difference between plant and animal cells is the fact that plant cells
do not have the capacity for movement.
 Movement of animal cells is important in normal cell function (e.g., phagocytic cells).
Movement is critical in development.
 Animal cells are also not able to trap energy from light since they lack chloroplasts.
They are required to get energy necessary to life by eating other living things.
COMPONENTS OF THE CELL

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Cell Membrane
The cell membrane separates the cell from its surroundings and
comprises a double molecular [Link] has an "oily" interior while
both surfaces are attracted water. The cell membrane is effectively
continuous with the endoplasmic reticulum and the nuclear
membrane. The cell membrane at the cell surface has many proteins
associated with it. Some proteins act as receptors for molecules
outside the cell, whilst others transport materials across the membrane.
Name(s): cell membrane, plasmalemma, plasma membrane
Location: the outer surface of the cell but frequently convoluted and is effectively continuous
with ER and nuclear envelope.
Appearance: double molecular layer.
Size: about 6-7nm in depth (excluding associated proteins).
Function: segregation of cell from surroundings and control of traffic in and out of the cell.

Nucleus, Nucleolus, Nuclear Envelope and Nuclear Pores

The nucleus sits roughly in the middle of the cell and contains the cell's
genetic information encoded in DNA. The nucleus is demarked by a
double membrane, the nuclear envelope, which segregates the nuclear
contents from the rest of the cell. Molecular portals, called nuclear
pores, permit certain traffic in and out of
the nucleus.
Name(s): nucleus
Location: approx centre of cell
Appearance: usually spherical or ovoid
but may be lobed
Size: usual range: 5 - 10 micrometers
Function: contains the genetic information of the cell coded in
DNA
The nucleolus is where the components of ribosomes are
manufactured. These ribosomal components exit through the
nuclear pores and enter the cytoplasm where they assemble into
ribosomes.
Name(s): nucleolus
Location: roughly in centre of nucleus.
Appearance: approximately spherical but with an ill-defined edge
Size: about 1 micrometer in diameter
Function: production of robosomal components
The nuclear envelope is a double-layered membrane that separates
the interior of the nucleus from the rest of the cell. It is bridged by
numerous nuclear pores and its outer layer is continuous with the
membrane of the rough endoplasmic reticulum.
Name(s): nuclear envelope, nuclear membrane
Location: surrounds nucleus
Appearance: double membrane, punctuated by numerous nuclear

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pores and with attached ribosomes

Size: total about 40nm in depth
Function: segregates nucleus from rest of cell
The nuclear pores are the rosette-like purple structures that are scattered over the nuclear
envelope.
Name(s): nuclear pore (the actual 'gap"), nuclear pore complex (gap + surrounding protein
machine)
Location: all over nuclear envelope.
Appearance: flower-like on surface. 8-fold structure. Complex structure in and beneath nuclear
envelope.
Size: about 120nm in diameter.
Function: controls transport in and out of nucleus.

DNA BASICS
The nucleus contains the genetic information in the form of chromatin, highly folded ribbon-
like complexes of deoxyribonucleic acid (DNA) and a class of proteins called histones. When
a cell divides, chromatin fibers are very highly folded, and become visible in the light
microscope as chromosomes. During interphase (between divisions), chromatin is more
extended, a form used for expression genetic information. The DNA of chromatin is wrapped
around a complex of histones making what can appear in the electron microscope as "beads on
a string" or nucleosomes. Changes in folding between chromatin and the mitotic chromosomes
is controlled by the packing of the nucleosome complexes. DNA or deoxyribonucleic acid is a
large molecule structured from chains of repeating units of the sugar deoxyribose and phosphate
linked to four different bases abbreviated A, T, G, and C. DNA contains the information for
specifying the proteins that allow life. The process of mitosis is designed to insure that exact
copies of the DNA in chromosomes are passed on to daughter cells.
Mitochondria
A mitochondion appears as an elongate ovoid body. The
mitochondrion's double membrane can just be discerned. The vertical
bulkhead-like structures are cristae, formed from infoldings of the inner
membrane. These cristae are the site of much enzymic activity involved
in energy production for the cell. Mitochondria are thought to have
been free-living organisms that became incorporated into cells early on in evolution. They even
contain their own DNA, just like a separate life-form. Such mitochondrial DNA is very useful
in constructing family trees.
Name(s): mitochondrion
Location: scattered throughout cytoplasm
Appearance: long ovoid with a double membrane, the inner one infolds to create bulkhead-like
structures called cristae.
Size: variable in range of several micrometers
Function: energy metabolism
Endoplasmic reticulum

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The rough endoplasmic reticulum is seen as stack of cisternae

connected by bridges. It has an interior space or "lumen" which is
continuous with the lumen of the nuclear envelope. The surface is
covered by ribosomes, which impart the "rough" appearance. Proteins
synthesised by these attached ribosomes pass into the lumen. Small
transitional vesicles bud from the roughER which carry proteins
destined for processing by the Golgi. As well as connecting to the nuclear envelope, the rough
ER is continuous with the smooth endoplasmic reticulum.
Name(s): rough endoplasmic reticulum, rough ER
Location: throughout cytoplasm
Appearance: sacs
Size: about 100nm in depth
Function: used in protein synthesis.
The smooth endoplasmic reticulum is seen as a tangle of
interconnected tubules. Unlike the rough ER, it lacks ribosomes. The
smooth ER is continuous with the rough ER but has different functions.
Name(s): smooth endoplasmic reticulum, smooth ER, tubular
endoplasmic reticulum, agranular endoplasmic reticulum
Location: in cytoplasm
Appearance: interconnected and ramifying tubules; no ribosomes
Size: tubules are about 150nm in diameter (variable)
Function: used in lipid synthesis, detoxification and other metabolic processes.
Golgi Complex
Golgi complex is seen as a stack of membranous sacs tilted at 45
degrees and receives proteins synthesised in the rough endoplasmic
reticulum. These are ferried to the Golgi by transfer vesicles. The
proteins are then processed by the Golgi for export, membrane use or
for inclusion in lysosomes.
Name(s): Golgi complex, Golgi apparatus, Golgi stack, Golgi body,
Golgi
Location: variable
Appearance: stack of discoid saccules but may form complex network. Surrounded by
vesicles.
Size: variable but approx 2500nm across
Function: processing of proteins
Centrioles
The centrioles are two tube-like objects seen very close
to the nucleus. Centrioles are bundles of microtubules
that sit in a grainy region called the centrosome. From
the edge of this region, microtubules assemble and
project in ray-like fashion to the edges of the cell. Their
central location gives the centrioles (and centrosome)
their names.
Name(s): centrioles
Location: near nucleus at approximate centre of cell.

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Appearance: tubular bundle of microtubules. There are 2 centrioles in the centrosome.

Size: about 500nm in length
Function: associated with the centrosome that generates microtubules. During cell division, the
centrioles go to opposite sides of the cell and organise the microtubules that drag the
chromosomes apart (so that one set each of the duplicated chromosomes end up in each
daughter cell).
Microtubules
Microtubules are spoke-like structures that originate near the centre of the cell at the
centrosome. The centrosome contains two centrioles, which are bundles of microtubules.
Microtubules are protein polymers which are relatively rigid and afford the cell some strength.
Microtubules also act in a funicular-like way to help objects move around in the cell.
Name(s): microtubule
Location: throughout cell radiating from centromere
Appearance: slender, individual, spoke-like.
Size: about 25nm in diameter
Function: cell support, movement of cell structures (including the pulling apart of daughter
chromosomes in cell division).
Lysosomes
Lysosomes originate from the trans surface of the Golgi and they contain a
variety of powerful enzymes (hydrolases) that break down
material. Prions (the rogue proteins associated with Mad Cow Disease) are
resistant to degradation by lysosomal enzymes and so accumulate in the
cell.
Name(s): lysosome
Location: usually towards periphery of cell near Golgi.
Appearance: approximately spherical with a single membrane.
Size: variable; generally in the range: 200 - 400 nm
Function: contain powerful digestive enzymes that are used to destroy invading matter and
unwanted cellular material.
Ribosomes
Ribosomes are bead-like objects that are attached to the exterior (cytoplasmic side) of the rough
endoplasmic reticulum. Ribosomes are concerned with protein synthesis. Stringing them
together is messenger RNA which directs protein manufacture as it passes through the
ribosomes. The growing proteins project into the cavity (lumen) of the rough ER. Other
ribosomes are free in the cytoplasm. Such membrane-bound ribosomes impart a beaded (rough)
appearance to endoplasmic reticulum when it is inspected by an electron microscope.
Name(s): bound ribosomes, attached ribosomes
Location: the outer (cytosol) surface of the rough endoplasmic reticulum.
Appearance: approximately spherical bodies often arranged in strings or spirals (because they
are strung together by messenger RNA)
Size: about 25nm in diameter
Function: synthesis of proteins
Polysomes are variable in length and are strings of ribosomes joined by messenger RNA. As
the mRNA feeds through these ribosomes, so proteins are synthesised. The proteins synthesised

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by these free ribosomes pass into the cytoplasm. (Proteins that are destined for the interior of
the rough endoplasmic reticulum have a lead sequence that binds to the rough ER. This links
their associated ribosomes to the surface of the rER.
Name(s): polysomes, polyribosomes
Location: free in cytoplasm
Appearance: approximately spherical bodies often arranged in strings or spirals (because they
are strung together by messenger RNA).
Size: ribosomes are about 25nm in diameter
Function: synthesis of proteins
Peroxisomes
Unlike lysosomes, peroxisomes do not bud from the Golgi but seem to
originate independently. Like mitochondria, they may once have been
separate organisms that became incorporated into cells early on in evolution.
They oxidise various materials and then dispose of the hydrogen peroxide
that results using an enzyme called catalase.
Name(s): peroxisome
Location: in cytoplasm
Appearance: variable; approx spherical with a single membrane and a granular or crystalline-
like interior in some cases.
Size: variable (illustrated at about 700nm in diameter).
Function: oxidise materials and then catalyse the destruction of the resulting hydrogen
peroxide.
CELL CYCLE
The cell cycle is an ordered set of events, culminating in
cell growth and division into two daughter cells. Non-
dividing cells not considered to be in the cell cycle. The
stages are G1-S-G2-M. The G1 stage stands for "GAP 1".
The S stage stands for "Synthesis". This is the stage when
DNA replication occurs. The G2 stage stands for "GAP
2". The M stage stands for "mitosis", and is when nuclear
(chromosomes separate) and cytoplasmic (cytokinesis)
division occur.

Regulation of the cell cycle

How cell division (and thus tissue growth) is controlled is
very complex. The following terms are some of the features that are important in regulation,
and places where errors can lead to cancer. Cancer is a disease where regulation of the cell
cycle goes awry and normal cell growth and behavior is lost.
 Cdk (cyclin dependent kinase, adds phosphate to a protein), along with cyclins, are
major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to
M.
 MPF (Maturation Promoting Factor) includes the CdK and cyclins that triggers
progression through the cell cycle.
 p53 is a protein that functions to block the cell cycle if the DNA is damaged. If the
damage is severe this protein can cause apoptosis (cell death).

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o p53 levels are increased in damaged cells. This allows time to repair DNA by
blocking the cell cycle.
o A p53 mutation is the most frequent mutation leading to cancer. An extreme case
of this is Li Fraumeni syndrome, where a genetic a defect in p53 leads to a
high frequency of cancer in affected individuals.
p27 is a protein that binds to cyclin and CdK blocking entry into S phase. Recent research (Nat.
Med.3, 152 (97)) suggests that breast cancer prognosis is determined by p27 levels. Reduced
levels of p27 predict a poor outcome for breast cancer patients.
MITOSIS

Mitosis is nuclear division plus cytokinesis, and produces two identical daughter cells during
prophase, prometaphase, metaphase, anaphase, and telophase. Interphase is often included in
discussions of mitosis, but interphase is technically not part of mitosis, but rather encompasses
stages G1, S, and G2 of the cell cycle.
Interphase
The cell is engaged in metabolic activity and performing its prepare for mitosis
(the next four phases that lead up to and include nuclear division).
Chromosomes are not clearly discerned in the nucleus, although a dark spot
called the nucleolus may be visible. The cell may contain a pair of centrioles (or
microtubule organizing centers in plants) both of which are organizational sites
for microtubules.
Prophase
Chromatin in the nucleus begins to condense and becomes visible in the light
microscope as chromosomes. The nucleolus disappears. Centrioles begin
moving to opposite ends of the cell and fibers extend from the centromeres.
Some fibers cross the cell to form the mitotic spindle.

Prometaphase
The nuclear membrane dissolves, marking the beginning of prometaphase.
Proteins attach to the centromeres creating the kinetochores. Microtubules
attach at the kinetochores and the chromosomes begin moving.

Metaphase
Spindle fibers align the chromosomes along the middle of the cell nucleus. This
line is referred to as the metaphase plate. This organization helps to ensure that
in the next phase, when the chromosomes are separated, each new nucleus will
receive one copy of each chromosome.

Anaphase
The paired chromosomes separate at the kinetochores and move to opposite
sides of the cell. Motion results from a combination of kinetochore movement
along the spindle microtubules and through the physical interaction of polar
microtubules.

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Telophase
Chromatids arrive at opposite poles of cell, and new membranes form around
the daughter nuclei. The chromosomes disperse and are no longer visible under
the light microscope. The spindle fibers disperse, and cytokinesis or the
partitioning of the cell may also begin during this stage.

Cytokinesis
In animal cells, cytokinesis results when a fiber ring composed of a protein
called actin around the center of the cell contracts pinching the cell into two
daughter cells, each with one nucleus. In plant cells, the rigid wall requires that
a cell plate be synthesized between the two daughter cells.

CHAPTER TWO

TISSUES

2.0 Introduction

In succinct terms, collection of specialized cells that perform limited number of functions is
called a tissue. The human body is composed of four basic tissues:

o Epithelium
o Connective Tissue
o Muscle Tissue
o Nerve Tissue

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2.1 EPITHELIUM

INTRODUCTION TO THE CONCEPT OF EPITHELIUM

Single celled organisms have very little control of their internal environment. If a group of
single-celled organisms become linked together to form a multicellular colony, then there is the
possibility for mutual cooperation of cells involving cellular differentiation and specialization for
specific functions. The cells at the exterior of the colony can be modified to assume functions
related to their position close to the external environment, whereas the cells deeper in the colony
can assume different functions. This specialization of function provides added advantages to the
multicellular organism. The layer of cells near the periphery can be modified for protection or
defense and can prevent fluid loss or desiccation. These cells can also be modified for metabolic
purposes such as control of substances taken up by the organism or excreted and for the
collection of information or signals from the external environment. These cells at the border
between the external and internal environments, the epithelial cells, are extremely important in
many aspects of physiological homeostasis.

2.2 FEATURES OF EPITHELIUM

Epithelium lines the surfaces of the body and is mainly located on the borders between the
external and internal environments. Epithelium also lines all the internal body spaces that have a
connection with the external environment at some stage.

Epithelium plays an important role in:

 Homeostasis of the body

 Maintaining the physiological parameters of the internal environment different from
those outside the body.

Epithelium is a tissue composed of cells, tightly-bound to each other, with no intercellular

connective tissue. There are specializations of the cell membranes that play roles in maintaining
the integrity of the tissue.

Epithelium is an avascular tissue and has no integral blood supply.

Epithelium develops in the embryo from all the three germ layers (Ectoderm, Mesoderm,
Endoderm). For example, the epidermis of the skin is ectodermal in origin, the epithelium lining
the serous cavities (peritoneum, pleura, pericardium) is derived from mesoderm (and is often
referred to as mesothelium), whereas the epithelium lining most of the intestinal tract is
endodermal. The endothelium, lining the blood vessels, is not a true epithelium, as it is derived
from mesenchyme (and should be considered as belonging to connective tissue. Moreover,
endothelium has no connection at any stage with the external environment.)

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2.3 FUNCTIONS OF EPITHELIUM

Owing to the strategic location of epithelium at the border between the internal and external
environments, the functions of epithelium are many and varied, but can be conveniently divided
into two major categories: protective or metabolic.

Protective functions of epithelium include protection against:

 mechanical damage
 loss of fluids (desiccation) - waterproofing
 invasion of foreign bodies

Metabolic functions of epithelium include:

 Exchange of metabolites, typically described as ion-transport. All the substances

entering or leaving the body must pass through epithelium and are under its control. The
ion-transporting epithelium may become highly specialized for absorption or excretion.

 The glandular secretions of the body by glands (exocrine and endocrine) are mainly a
function of specialized epithelium.

 Some epithelia are modified for sensory reception including recognition of sensory
stimuli such as pain or as chemoreceptors (such as taste buds).

Polarity

Epithelial cells are polarized cells and we can distinguish different areas of the cells (apical,
basal, lateral) with specific structural modifications (unlike other tissues, where structural
polarity is not found).

Specific structures found on the apical surface (the free surface facing the lumen or external
environment) include: microvilli, stereocilia, cilia or flagella.

The lateral surfaces (between adjacent epithelial cells) typically have "junctional complexes"
including:

 tight junctions (impermeable, enabling the organism to maintain the integrity of its
internal environment)
 adhering junctions (desmosomes) promoting adhesion and reinforcing the structural
integrity and sites for stress fibers
 communicating junctions (gap junctions or nexuses), which allow the exchange of
nutrients, ions, signals between adjacent cells).

Epithelial cells are separated from the underlying connective tissue by a basal lamina, secreted
by the cells themselves. The plasmalemma at the base of epithelial cells, especially those with

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metabolic function (ion-transporting epithelia) may be modified by having marked invaginations

to increase the surface area.

2.4 MORPHOLOGICAL CLASSIFICATION OF EPITHELIA

Epithelia are described according to the number of layers they possess and the appearance of the
cells at the border adjacent to the external environment.

a) Simple epithelia are composed of a single layer of epithelial cells.

b) Stratified epithelia are composed of more than one layer of epithelial cells.

The histological appearance describing the various epithelia is that seen in vertical sections. It is
customary when drawing epithelia to show the free surface directed to the top of the page,
whereas the basal surface is depicted in the direction of the bottom of the page.

1) SIMPLE EPITHELIA

Simple epithelia consist of a single layer of cells.

When seen in vertical sections the epithelial cells are described as :

 squamous (flattened)
 cuboidal (more square or cube-like)
 columnar (tall and thin)

If all the cells in the epithelium consist of a single cell type, the epithelium is described as being
homogeneous. If the epithelium has more than one cell type, the epithelium is described as being
heterogeneous.

If the apical edge of the epithelium has cilia, the epithelium is described as being ciliated.

Epithelial cells that have large numbers of microvilli on the apical or luminal surface (such as the
columnar absorptive cells of the small intestine) are described as possessing a brush border.

If histological sections of the simple epithelium are cut tangentially, they may give a false
impression of being composed of more than one layer (stratified).

EXAMPLES OF SIMPLE EPITHELIA

a) Simple squamous epithelia line the serous cavities of the body (peritoneum, pleura,
pericardium). These epithelia are also known as Mesothelia (because of their mesodermal
origin).

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In vertical sections these simple squamous epithelia are seen as very flat, elongated cells.
Characteristics

-thin, foundin protected regions (locations:mesothelium(serosa), endothelium(blood vessels,

heart), kidney tubules, alveoli of lungs)

-Functions:absorption, diffusion, filtration orsecretion

Fig 1: Simple squamous epithelia

b) Simple cuboidal epithelia line many small ducts in the body. Examples include the urinary
ducts of the kidney, the bile ductules of the liver, or the cells lining thyroid follicles. Functions:
secretion or absorption

Fig 2: Simple Cuboidal epithelia

C) Simple columnar epithelia: This epithelium hasnuclei line up near basal lamina, and the
apical surface of cells often has microvilli “brush border” (in intestine) and some have goblet
[Link]: line the gallbladder, the larger ducts near the urinary papilla of the renal pyramids
and the absorptive cells of the small intestine. Absorption or secretion is the principal functions.

Fig 3: Simple columnar epithelia

Ion-transporting (metabolic) epithelia are typically simple epithelia.

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Fig 4: Cross sectional diagram of squamous, stem cells and connective tissues

2) STRATIFIED EPITHELIUM

Stratified epithelia consist of more than one layer of cells. These typically are at sites needing a
more defensive, rather than a metabolic function.

Stratified epithelium is described as squamous when the cells nearest to the external environment
are flattened. In typical multilayered epithelium, the cells nearest to the base of the epithelium
are more columnar or cuboidal, whereas the cells nearer to the surface are flatter. The more basal
areas are sites of cell proliferation (mitosis) and there is a continuous movement of cells in the
direction of the free surface, with the flattened surface cells being sloughed off.

i) Stratified squamous epithelium

Found in the esophagus and epidermis of the skin. The stratified epithelium of the skin
(epidermis) has a layer of keratin and is called a keratinized (or dry) [Link]:
provide protection from abrasion,pathogens, and chemicals.

Non-keratinized (mucosa)-kept moistLocations:, mouth, esophagus, anus, vagina

ii) Stratified Cuboidal Epithelium

This epithelium is rare, but is however found in sweat and mammary glands where they function
in absorption and secretions.

iii) Stratified Columnar Epithelium

This epithelium is rare, and usually in two or multiple layers with only apical layer columnar.
Locations (tiny parts of): pharynx, epiglottis, anus, mammary glands, salivary glands, urethra

Functions: minor protection

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Fig 5:Stratified squamous cuboidal columnar epithelia

Pseudostratified columnar epithelium

Some epithelia give the false appearance of being stratified, but in effect consist of a single layer
of irregular columnar cells, all in contact with the basal lamina. Pseudostratified columnar
epithelium is the typical epithelial type of much of the respiratory tract. This epithelium also is
ciliated and is an example of a heterogeneous epithelium owing to an additional cell type
(mucus-secreting unicellular goblet cells). One additional characteristic feature of
pseudostratified epithelium is that the nuclei of adjacent cells are not orderly arranged but appear
at different levels.

Characteristics:

 several cell types:

 varying shapes and functions
 all cells contact basal lamina
 some too short to reach apical surface
 nuclei scattered so it appears stratified
 tall cells have cilia on apical surface
 goblet cells (mucus) often present

Location: nasal cavity, trachea, bronchi, male reproductive tract, female uterine tubes

Function: move material across surface

Fig 6: Structure of a pseudostratified columnar epithelium

Transitional epithelium

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Transitional epithelium is a form of stratified epithelium lining the urinary bladder and part of
the urinary tract. This epithelium is subjected to large mechanical changes and can adapt
accordingly. If the bladder is empty, the epithelium appears to be thicker and have more layers,
than when the bladder is full and distended, in which case, the cells appear more stretched.
Transitional epithelium can be recognized by the large rounded epithelial cells lining the lumen
(in contrast to other stratified epithelia, where such surface cells are squamous).

Functional: to withstand stress

Figure 7: Structure of relaxed and stressed transitional epithelia

Basal lamina

All epithelia lie on a basal lamina, separating them from the underlying connective tissue
(lamina propria). The basal lamina provides structural support and acts in part as a selective
barrier for the epithelial layer. The basal laminae are formed by the cells themselves. In some
cases the basal laminae are greatly thickened (as in the glomeruli and filtration system of the
kidneys). (The older term "basement membrane" should be avoided). The basal laminae, as seen
by transmission electron microscopy, are seen to be formed from an electron-dense layer (50-80
nm thick) composed of non-fibrous type IV collagen and the proteoglycan, heparan sulfate,
surrounded on both sides by a less-dense layer containing the glycoprotein, laminin.

Basal laminae are not exclusive features of epithelia, but are also found associated with
endothelia and some other cell types.

The border between epithelia and the underlying connective tissue is sharp and distinct. In the
case of stratified epithelium this border may be ridged or consist of papillae.

Epithelia are avascular. Blood vessels of the underlying connective tissue (lamina propria)
supply the necessary nutrients and metabolites, which are transported to and from the epithelial
cells by diffusion.

Some epithelia, especially the stratified epithelia of the epidermis and the nasal mucosa may
have sensory nerve endings. These help transmit information from the external environment.

Proliferation and regeneration

Epithelia are present in vulnerable sites of the body, where they are continually exposed to the
hazards of the external environment. Epithelial cells are constantly subjected to mechanical
damage, destroyed, or sloughed off. Epithelia have remarkable proliferative properties and

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typically show many dividing cells (mitoses) in order to replace the cells lost and maintain the
integrity of the tissue. In cases of trauma or wounds, the epithelia need to cover the lesion as
rapidly as possible, repair the lining tissue and prevent damage to the underlying tissues.

Most of the cancers of the body are the result of uncontrolled proliferation of epithelial cells
(aden).

GLANDULAR EPITHELIUM

One of the specialized functions of epithelia is secretion.

Glands consist of groups of epithelial cells modified to synthesize and secrete.

Glands are classified as:

i) Exocrine glands, which secrete to the external environment via ducts.

 “internally secreting “hormones” into interstitial fluid = blood

They regulate and coordinate activitiese.g. pancreas, thyroid, thymus, pituitary

ii) Endocrine glands, which secrete directly into the blood (ductless glands): “externally
secreting” hormones into duct = epithelial surfacee.g. digestive enzymes, perspiration,tears,
milk, mucus

iii)Mixed glands, which have both exocrine and endocrine secretion.

Secretion of exocrine glands is classified as:

(a) Merocrine (or eccrine) secretion

This is the most common type of exocrine secretion. Secretory granules (packaged in the Golgi
bodies) migrate to the apical surface of the cell. The membranes of the secretory granules fuse
with the apical membrane and the secretion is released to the external environment by
exocytosis.

(b) Apocrine secretion

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With apocrine secretion the apical portion of the cells, together with the secretory contents, are
budded off and released to the lumen or external environment. Examples of such apocrine
secretion are found in the apocrine sweat glands of the armpits.

(c)Holocrine secretion

Holocrine secretion involves the secretion of whole cells and their contents. This is best seen in
the sebaceous glands associated with hairs of thin skin.

CHAPTER THREE

CELL JUNCTIONS/CONNECTIVE TISSUES

Introduction
 A cell junction (or intercellular bridge orintercellular connections) is a type of
structure that exists within the tissue of some multicellular organisms, such as animals.
Cell junctions consist of multi-protein complexes that provide contact between
neighbouring cells or between a cell and the extracellular matrix. They also build up the
paracellular barrier of epithelia and control the paracellular transport. Cell junctions are

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especially abundant in epithelial tissues. Examples include tight, epithelial, gap junctions,
adherens junctions, desmosomes and hemidesmosomes (anchoring junctions)

Fig 6: Tight junction between cells.

Fig 7: Gap junction between cells.

3.2 CONNECTIVE TISSUES

It is important to note that connective tissues (collection of cells), that are usually vascularized,
consists of cells in a matrix (a mixture of fibers and ground substance) and never exposed to the
environment. In brief the major components of connective tissues: specialized matrix-producing,
extracellular fibers and ground substances ( gel fluids consisting of glycosaminoglycan).
3.2.1 Functions of connective tissues
 Establish structural framework
 Transport fluid and dissolved materials
 Protect organs
 Support, surround, interconnect tissues
 Store energy reserves

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 Insulate body
 Defend against pathogens

3.2.2 Classification of Connective tissues

1. Connective Tissue Proper:many cell types and fiber types in thickground substance. There
are two types of connective tissue proper.
a) Loose connective tissue: open fiber framework
b) Dense connective tissue: tightly packed fibers, mostly found in joints, ligaments, cartilages.
2. Fluid Connective Tissue:many cell types in watery matrix withsoluble fibers
3. Supporting Connective Tissue:limited cell population in tightly packedmatrix
1- Connective Tissue Proper

3.3 Understanding the architectural make-up of loose connective tissues:

 viscous ground substance
 varied extracellular fibers
 varied cell population
 Ground substance: rich in GAGs
 (prevents microbe penetration)

Students studying histology are always told to place a degree of emphasis on the types of fibers
in connective tissues, since they play a vital role in the architectural make-up of these tissues
especially providing support.

A) Fiber types:
1. Collagen fibers: collagen protein
 rope like, long, straight
 resists force
 The most commonly found fiber

2. Reticular fibers: collagen protein

 branchy, forms framework
 framework of an organ = stroma

3. Elastic fibers: elastin protein

 wavy, flexible
 designed to stretch

B) Cell types:
1. Fibroblasts
 These are the most common and most abundant cell types
 They responsible for the secretion of ground substance:GAGs
 secrete fiber proteins (collagen, elastin)
 some specialized types:These some names of fibroblast found in certain organs;

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a) chondrocytes (cartilage)
b) osteocytes (bone)

2. Mesenchymal cells
 stem cells
 They differentiate to replace CT(connective tissue) cells afterinjury (e.g. fibroblasts,
adipocytes)

3. Adipocytes (fat cells)

 These cells store triglycerides
 Their organelles peripheral nature
 number, size and location of cells varies

4. Macrophages
 phagocytic for defense
 some fixed in tissues
 others migrate from blood to tissuesafter injury

5. Microphages
 neutrophils and eosinophils
 phagocytic
 migrate from blood to site of injury

6. Lymphocytes: B and T cells

 involved in immune response
 make antibodies, attack foreign cells
 increase in number during infection constantly migrate between blood and tissues and
lymph

7. Mast cells
 contain histamine and heparin
 stimulate inflammation in response to injury

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Figure 8: Loose Connective tissue.

Some examples of loose connective tissues

1) Adipose Tissue
 This tissue is made up of 90% adipocytes
Locations:
 deep to skin
 surrounding eyeballs, kidneys, heart
Functions:
 padding and insulation
 Energy storage.

Fig 9: Structure of an adipose tissue (composed of mainly adipocytes)

2) Reticular Tissue
 stroma of organs
 consists of reticular fibers

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Locations: some organs:

E.g. lymph nodes, bone marrow, liver
Function: support parenchyma cells

Fig 10: Structure of areticular tissue (composed of mainly of reticular fibres)

3.4 Dense Connective tissues

These tissues are poorly vascularized, composed of mainly fibers, little ground substance and
only fibroblasts. There are three types of dense connective tissues:
 Dense regular CT
 Dense irregular CT
 Elastic CT

A) Dense regular CT
They are composed of bundles of parallel collagen fibers, aligned with direction of force.
Locations:
 tendons (muscle to bone)
 ligaments (bone to bone)
 muscle coverings
 fascia

Function:
 high strength attachment
-stabilize positions

Figure 11: Structure of a dense regular CT

B)Dense Irregular CT
This type of dense connective tissue is made up of mesh-network of collagen fibers.
Locations:
 capsules of organs & fascia
 periosteum (sheath around bone)

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 perichondrium (around cartilage)

 dermis (deep skin)

Function:
 resist tension from many directions
 attachment

Figure 12: Structure of a dense irregular CT

C) Elastic CT
This dense connective tissue is made of mostly elastic fibers and some collagen.
Locations:
 vertebral ligaments
 artery walls
Function:
 strength with stretch and flex

Figure 13: Structure of Elastic CT

2- Supporting Connective Tissue

These connective tissues are strong framework tissues with few cells in a fibrous matrix.
Functions:
 support and shape

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 mature cells in lacunae

Types of supporting tissues
A) Cartillage

Composition:
 Matrix: 80% water, firm gel of GAGs made of chondroitin sulfate and hyaluronic acid, +
fibers
 Cells: chondrocytes (in lacunae) (cells formed the matrix)
 no innervation
 avascular (antiangiogenesis factor)
 surrounded by perichondrium:outer layer = dense irregular CT for protection and
attachment
 Inner layer = cellular (fibroblasts) in charge of growth and repairs.

Types of cartilage:
1. Hyaline Cartilage
 matrix contains fine, closely packed collagen fibers
 tough, springy
Locations:
 ribs
 nose
 respiratory tract
 articular surfaces (no perichondrium)
Function:
 -provide stiff flexible support
 -reduce friction between bones

Fig 14: Structure of a hyaline cartilage

2. Elastic Cartilage
 matrix contains elastic fibers
 They are flexible
Locations:
 auricle of ear
 epiglottis
Function:
 resilient, flexible, shape holding support

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Fig 15: Structure of an elastic cartilage

3. Fibrocartilage
This cartilage’s matrix contains dense, tough, durable, interwoven collagenfibers with little
ground substance.
Locations:
 knee (meniscus)
 pubic symphasis
 intervertebral discs
Functions:
 resist compression
 absorb shock

Fig 16: Structure of a fibrocartilage

Bone / Osseous Tissue

Composition:
These are highly vascularized tissues with very little ground substances.
 matrix = 2/3 calcium salts for strength(calcium phosphate + calcium carbonate) and
1/3 collagen for flexibility to resist shatter
 cells = osteocytes(cells formed the matrix)
 Osteocytes are located in lacunae
 connected by cytoplasmic extensionsthat extend through canaliculi
 canaliculi are canals necessary for nutrient nourishment and wasteexchange, no
diffusion through calcium
 Surrounded by periosteum:outer fibrous layer for attachmentinner cellular layer for
growth and repair.
Location:
Bones
Functions:

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 support and protection

 levers for movement
 storage of minerals

Fig 17: Structure of a bone tissue.

3.5 BONE CELLS

Because bone is made up of minerals and is hard, many people think that it is not living material.
But a bone in a living animal consists of both living tissue and non-living substances. Within
the "alive bone" are blood vessels, nerves, collagen, and living cells including fourdifferent cell
types are found in developing and developed bones:

 Osteoprogenitor cells
 osteoblasts (cells that help form bone),
 Osteocytes
 Osteoclasts (cells that help eat away old bone).

Osteoprogenitor cells
Bone, like other connective tissue in the embryo, is derived from mesenchyme cells. In the
foetus, most of the skeleton is made up of cartilage, a tough, flexible connective tissue that has
no minerals or salts. As the foetus grows, osteoblasts and osteoclasts slowly replace cartilage
cells and ossification [Link] birth, flattened, poorly-differentiated, mesenchyme-like cells
are found in the periosteum and endosteum. These cells can divide (mitosis) and differentiate
into bone cells (osteogenic potential) and as a result are known as osteoprogenitor cells.

Osteoblasts
The first cells to develop from the osteoprogenitor cells are the osteoblasts. Osteoblasts are
involved in the formation of bone and are found on the boundaries of developing and growing
bone. The cells are typically oval, with a large eccentric nucleus, and the cytoplasm is fairly
basophilic. These cells are very active in synthesizing and secreting the components of the bone
matrix and have well-developed rough endoplasmic reticulum (RER), Golgi bodies and granules.
Osteoblasts are rich in the enzyme alkaline phosphatase, which plays a major role in the

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formation of the mineral deposits in the matrix. The collagen fibers are synthesized and secreted
by the osteoblasts.
The matrix closest to the osteoblasts is not yet calcified and is known as osteoid or prebone.
This osteoid is rich in collagen fibers. Small membrane-bound matrix vesicles (not visible by
light microscopy) are budded off processes of the osteoblast cell membrane and secreted to the
matrix. These play an important role in the calcification process of the matrix.

Osteocytes
Osteocytes are mature bone cells that develop from osteoblasts and are located in lacunae
within the bony matrix. Osteocytes have cytoplasmic processes located in canaliculi, which
penetrate the bony matrix. Cytoplasmic processes from one osteocyte make contact with the
processes from neighboring osteocytes and can communicate via gap junctions. Because the
bony matrix is calcified there is no possibility of diffusion except via the network of canaliculi.

Osteoclasts
Osteoclasts are the largest of the bone cells (20-100mm diameter) and are multinuclear (with up
to 50 nuclei). Osteoclasts are involvedin bone resorption and can be found on the eroding
surfaces of bone, often in cavities known as Howship's lacunae. The osteocytic cell membrane
closest to the bone undergoing resorption has multiple invaginations and is known as the
"ruffled border". The cells are metabolically very active, possess large numbers of
mitochondria (resulting in the acidophilia of regular staining) and have well-developed Golgi
bodies. Osteocytes synand secrete the enzyme acid phosphatase, which is involved in the
erosion of the bony matrix.
Osteoclasts originate from monocytes and are included in the mononuclear phagocyte system.

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CHAPTER FOUR

EXAMINATION OF TISSUE IN A HISTOPATHOLOGY LABORATORY

4.0 Introduction
The understanding of tissue is important in the histopathology laboratory especially anatomic
details of complex specimens must be well understood before processing of the tissue begins.
Tissue examination is important too in making educated judgments about the extent of the
tissue’s condition and need for further resection if need [Link] is the process by
which pathology specimens are inspected macroscopically (unaided eye to) obtain diagnostic
information, while being processed for further microscopicexamination.
Individuals trained in histology and cytologyare often able to gather diagnostically critical
information in this stage of processing, including the stage and margin status of surgically
removed tumors.

4.1 Examination of fresh specimens.

The first step to be done when any specimen is received at the histopathology unit is to
a) Verify specimen labeling and patient identification, if okay you then proceed to give a
unique code.
b) Verify that the tissue section is from a documented anatomical site from which the
specimen was obtained.
c) Note time of reception of specimen
d) Check if the container has a fixative in the right quantity and check for leakages.

e) Examine and Palpate All External Surfaces of the Specimen Carefully: Understanding the
anatomy of the specimen, examine and palpate all surfaces noting the following:

 Colour and texture (consistency)

 Presence of nodules and defects
 Adherent tissues, marking sutures

Note: Insufficient clinical data should be communicated by the histo-technician to the

pathologist/cytologist in order to guide the appropriate diagnostic examination and interpretation
of the specimen.

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CHAPTER FIVE

Tissue Processing

5.1 Fixation of tissues

The process of tissue fixationis one the key important step in the histopathology laboratory.
The purpose of the fixative is to stabilize the protein in the tissue (whether biopsy or autopsy).
Once the tissue is removed from the body it will go through a process of self-destruction. This
process is known as Autolysis – which begins immediately after the cell death creating an
enzyme attack, which in place causes the breakdown of protein and eventual liquefaction of the
cell.
The enzymes appear to reverse their action; instead of synthesizing amino acids into cell
proteins, they begin to split proteins into amino acids. These amino acids diffuse out of the cells,
and as a result proteins no longer are coagulable by chemical reagents. All these actions are
called post-mortem changes.
The process of autolysis is more severe in enzyme-rich tissues, such as the liver, brain and
kidney, and is less rapid in tissues such as elastic fibers and collagen. By light microscopy,
autolyzedtissue presents a `washed-out' appearance with swelling of cytoplasm, eventually
converting to a granular, homogeneous mass which fails to take up stains. If tissue is left without
any preservation, then a bacterial attack will occur (putrefaction).

With this at the back of our minds, a fixative is any substancethat is usedto preserve cells and
tissue constituents in as close a life-like state as possible and to allow them to undergo
further preparative procedures without change.A Fixative will thereforearrest autolysis and
bacterial decomposition and stabilizes the cellular and tissue constituents so that they withstand
the subsequent stages of tissue processing.
Fixation should also provide for the preservation of tissue substances and proteins, therefore, it is
the first step and the foundation in a sequence of events that culminates in the final examination
of a tissue section.

It is also important for future histotechnicians, some fixatives target overall tissue structure to,
these are called microanatomical fixatives. Those that target cellular components are called
cytological fixatives. Of this group, some are nuclear preservatives in contrast to others which
are far better at cytoplasmic preservation. Some other fixatives target particular chemicals in the
sectioned tissue, including enzymes and antigens. These are known ashistochemical fixatives.

A good fixative should be able to:

1. Penetrate rapidly to prevent post-mortem changes.
2. Coagulate cell contents into insoluble substances.
3. Protect tissue against shrinkage and distortion during dehydration, embedding and
sectioning.
4. Allow cell parts to become selectively and clearly visible by means of dyes and
improved refractive indices. This is the ability of a fixative to act as a mordant (assisting
in the attachment of dyes and proteins to each other during the staining process).
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5.2 Actions of Fixatives

A. Methods of Stabilizing Proteins

i) Physical Method
1. Heat (Physical method): This method is becoming more used in the histology laboratories
with the introduction of microwave fixation.
2. Desiccation (Physical method): Rarely used in histology. Touch preparations for Giemsa (It’s
an important stain the cytology laboratory) in the stains are probably the most common used for
this method.

ii)Chemical method
This is considered the primary method of fixation. These chemical reagents are used and may be
classified as:
 Additive – they chemically link or bind to the tissue and change it.
 Non-additive – include organic compounds such as acetone and alcohols, which act on
the tissue without chemically combining with the tissue. Ex: Methyl or Ethyl Alcohols
Among the frequently used chemicals are form aldehyde, ethyl alcohol, acetic acid, picric acid,
potassium dichromate, mercuric chloride, chromic acid and osmium tetroxide.

B. Coagulant and Non-Coagulant

1. Coagulation (coagulant) – will allow the solutions to penetrate into the interior of the tissue
very [Link] fixatives change the sponge-work of proteins into meshes through which
paraffin can easily pass, thus forming a tissue of proper consistency for sectioning. In addition,
the protein linkages are strengthened by coagulants against breaking down during later
procedures.
Disadvantage: coagulation tends to induce the formation of artificial structures (artifacts).

Coagulant Fixatives Reagents includes:

• Alcohol
• Zinc salts
• Mercuric chloride
• Chromium trioxide
• Picric Acid

2. Non-coagulant - they act by creating a gel barrier that makes solutions more difficult to
penetrate to the interior of the [Link]-coagulant fixatives produce fewer artifacts but if used
alone have the disadvantage of giving the tissue a poor con sistency for embedding.

• Formaldehyde
• Gluteraldehyde
• Osmium Tetroxide
• Potassium Dichromate
• Acetic Acid

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5.3 Some factors that affect fixatives

A. Temperature:
The factor influenced by temperature is the morphology of the tissue. Normally the fixation of
specimens for standard histology is carried out at room temperature for convenience. The use of
chemical reactions at higher temperatures to penetrate tissue, including those involved in the
fixation process will increase the rate of penetration of the fixative to the specimen; however, an
increase in temperature will also increase the rate of autolysis and diffusion of cellular
components.

B. Size
The penetration of fixatives into tissue is a relatively slow process and tissue blocks should either
be small or thin, in order to obtain satisfactory fixation. Large specimens should be opened and
washed of contents or sliced thinly before placement in fixative. Thick specimens will take a
very long time for fixatives to penetrate.

C. Time
In the histopathology lab, the tissue should be placed in fixative immediately after surgery, as
well as autopsies should be performed immediately after death. The more time it elapses between
interruption of the blood supply and fixation, the more post-mortem changes will be observed
under the microscope.

D. pH
The hydrogen ion concentration varies between fixatives, but in general, the pH should be kept
in the physiological range, between pH 4-9. If the pH of formalin is allowed to fall to a lower pH
this can produce formalin pigments. Even though in routine histology the pH is not usually
critical, in electron microscopy it is very important. The pH for the ultrastructural preservation of
great specimen the fixative should be buffered between 7.2 to 7.4.

5.4 Simple Fixatives used in the histopathology laboratory

It is important to note that that there is no universal fixative which will serve all requirements.
Each fixative has specific properties and disadvantages and their many different effects
emphasize the necessity for careful consideration and selection of the appropriate fixing reagent
when studies of specific cellular substances are planned.
a) FORMALDEHYDE
This is the most widely used fixative used in the histopathology laboratory and most especially
in our mortuaries. Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the
most widely employed universal fixative particularly for routine paraffin embedded sections. It is
a gas with a very pungent odor, soluble in water to a maximum extent of 40% by weight and is
sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid).
Precaution:Formaldehyde is an immediate irritant to the eyes, upper respiratory tract and the
skin, and safety precautions should include proper ventilation and exhaust, limited or restricted
exposure periods and thorough washing if spilt on tissue surfaces such as the skin.

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b) OSMIUM TETROXIDE
The most commonly used metallic ion in fixation is osmium tetroxide which was initially a
tissue fixative used in cytology, but poor penetration limited its application in light microscopy.
It is now largely employed as a secondary fixative in electron microscopy. Osmium tetroxide is
known to form cross-links with proteins as reflected in the rapid increase in viscosity of a protein
solution when they react together, however, there is very little additional information as to its the
mechanism of action.

c) ACETONE
Acetone is a clear, colorless, inflammable liquid which is miscible with water, ethanol and most
organic solvents. It has been used as a dehydrating agent in tissue processing and is more volatile
than alcohols and other dehydrants. It has a rapid action but causes brittleness in tissue if
exposure is prolonged and because it is volatile and inflammable, acetone is not used in
automated processing schedules.

5.5 Compound Fixatives used in the histopathology laboratory.

These are fixatives that on their own they cannot fix tissues, but can only function in
combination with another fixative or substances like salt.

A) ACETIC ACID
Acetic acid is never used alone but is often combined with other fixatives that cause shrinkage
such as ethanol and methanol. Acetic acid penetrates thoroughly and rapidly but lyses red blood
cells.

B) PICRIC ACID
Picric acid, when used in combination with other ingredients, leaves tissue soft and penetrates
well, precipitating all proteins. It will continue to react with the tissue structures and cause a loss
of basophilia unless the specimen is thoroughly washed following fixation.

5.6 MICROTOME

Microtome (the word is derived from the Greek wordmikros, meaning "small", and temnein,
meaning "to cut") is a tool used to cut extremely thin slices of material, known as
[Link] use steel, glass, or diamond blades depending upon the specimen being
sliced and the desired thickness of the sections being [Link] brief, microtomes are precision
instruments used to cut uniformly thin sections of tissue, supported in its embedding medium.
All microtomes have three main parts

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 Base( microtome body)

 Knife and its holder
 Block holder

Note: It is important for histology students to know that prior to sectioning or cutting by
microtomy, biological materials are usually placed in a more rigid fixative, in a process known
as embedding. This is achieved by the inflow of a liquid substance around the sample, such as
paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a "block"
which is readily cut. If this is not done, then cutting becomes very difficult.

5.6.1Types of microtomes

A. Sledge Microtome:

A sledge microtome is a device where the tissue to be sectioned is placed into a fixed holder
known as a shuttle, which then moves backwards and forwards across a knife. Modern sled
microtomes have the sled placed upon a linear bearing, a design that allows for the microtome to
readily cut many coarse sections. By adjusting the angles between the sample and the microtome
knife, the pressure applied to the specimen during the cut can be [Link] applications for
this design of microtome are of the preparation of large samples, such as those embedded in
paraffin for biological preparations. Typical cut thickness achievable on a sledge microtome is
between 1 and 60 µm.

[Link] microtome
This type of microtome is the most commonly used in the histology laboratory to section tissues.
This device operates with a staged rotary action such that the actual cutting is part of the rotary
motion.
In a rotary microtome, the knife is typically fixed in a horizontal position.
Through the motion of the sample holder, the sample is cut by the knife position 1 to position at
which point the fresh section remains on the knife. At the highest point of the rotary motion, the
sample holder is advanced by the same thickness as the section that is to be made, allowing for
the next section to be made.
The flywheel in many microtomes can be operated by hand. This has the advantage that a clean
cut can be made, as the relatively large mass of the flywheel prevents the sample from being
stopped during the sample cut. The flywheel in newer models is often integrated inside the
microtome casing. The typical cut thickness for a rotary microtome is between 1 and 60 µm. For
hard materials, such as a sample embedded in a synthetic resin, this design of microtome can
allow for good "Semi-thin" sections with a thickness of as low as 0.5 µm.

C. Cryomicrotome
This type of microtome is designed for the cutting of frozen samples; many rotary microtomes
can be adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup. The
reduced temperature allows for the hardness of the sample to be increased, which allows for the

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preparation of semi-thin [Link] is important that in this type of microtomy, the sample
temperature and the knife temperature must be controlled in order to optimise the resultant
sample thickness.

D. Vibrating microtome
The type of microtome operates by cutting using a vibrating blade, allowing the resultant cut to
be made with less pressure than would be required for a stationary blade. The vibrating
microtome is usually used for difficult biological samples. The cut thickness is usually around
30-500 µm for live tissue and 10-500 µm for fixed tissue. However, ideally the most appropriate
thickness for observation should range from 3-5µm.

E. Saw microtome
The saw microtome is especially designed to section hard materials such as teeth or bones. The
microtome of this type has a recessed rotating saw, which slices through the sample. The
minimal cut thickness is approximately 30 µm, and can be made for comparatively large
samples. As earlier mentioned, ideally the most appropriate thickness for observation of
histological/cytological sectionsshould range from 3-5µm.

F. Ultramicrotome

This microtome is special in that it can allow for the preparation of extremely thin sections, with
the device functioning in the same manner as a rotational microtome, but with very tight
tolerances on the mechanical construction. As a result of the careful mechanical construction, the
linear thermal expansion of the mounting is used to provide very fine control of the thickness.

5.6.2 Types of Microtome Blades

Microtome blades consist of a wedge of steel, glass,diamond, tungsten carbide or other hard
material normally having a maximum thickness of 11/2 inches which taper to a sharp edge. The
blades may normally have a width of up to 21/2 inches and a length of up to 20 [Link] edges
of the microtome knives must be frequently honed and/or stropped, the frequency dependent
upon the nature of the tissue sample being sectioned, and the knives must occasionally be
reground or reconditioned.
Microtome knife sharpening
A sharp knife/blade is essential for producing good tissue sections. Done by manual or automatic
methods, both involve two steps-honing and stropping.
 Honing refers to grinding the cutting edge of the knife on a hard abrasive surface to
sharpen the knife [Link] black vein, Arkansas, Yellow and Green belgian stones

 Stropping refers to the process of polishing an already fairly‟ sharp edge”

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5.6.3 Technique of Cutting

 Carefully insert the knife in the knife-holder and tightly screw it.
 Fix the block in the block holder.
 Move the block holder forward until the paraffin wax almost touches the knife
 Trim the excess wax: this is done by cutting the block by thickness of about 10-15μm till
the tissue surface is revealed
 Reset the thickness gauge to required thickness. 4-5 μrecommended for routine work.
 Subject the block surface to ice, in order to make the wax hard which would have become
soft by frictional heat.
 Begin cutting
 There should be a smooth continuous plastic flow of the sections in the form of a ribbon
 When the ribbon comes off it is held gently with a fine moistened brush or forceps and
then transferred to a slide.
 The slide is then dried on a hot plate set at a temperature of 55-60 oC and left for about 15
min.

5.6.4 Faults in paraffin section cutting.

Fault Possible Cause /reason Resolution/Remedy

1) Sections scored or cut Knife edge has small knicks sharpen
vertically
Knife is dirty clean

Calcium salts in tissue use softening solutions

2) Sections curl or roll up Knife is blunt sharpen

Tilt of knife is too great
3) Sections are alternatively Screws need tightening
thick and thin or each have
thick and thin zones
parallel to the knife edge.
Tissue is hard soften
Tilt of knife is too great
4)Sections crumble on Knife blunt sharpen
cutting
Wax too soft needs ice to be applied on
section
Wax crystallised due to slow
cooling or contamination
with water or clearing agent
5) Ribbons of section Block edges are not parallel Align the block edge parallel
curved to each other to blade.
Block edges not parallel to Align the block edge parallel
the knife to blade

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Wax too soft cool with ice

Blunt knife sharpen

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CHAPTER SIX

STAINS USED IN A HISTOLOGY LABORATORY

6.0 Introduction
Most staining reactions involve a chemical union between dye and stained substance through salt
linkages, hydrogen bonds, or others. Staining with these dyes results in a predictable colour
pattern based in part on the acid-base characteristics of the tissue. However, colour and colour
distribution are not absolutely reliable for discrimination between tissue components. Colour will
vary not only with specific stains used, but also with the conditions that exist during
preparation of the slide. These include everything from the initial fixing solution to the ionic
strength of the staining solution and the differentiating solvents utilized after staining.
Most histologic dyes are classified either as acid or as basic dyes. An acid dye exists as an anion
(negatively charged) in solution, while a basic dye exists as a cation (positive charge).

Any substance that is stained by the basic dye is considered to be basophilic; it carries acid
groups which bind the basic dye through salt linkages. When using hematoxylin, basophilic
structures in the tissue appear blue (or purple or brown; this varies according to the stain that is
being used).
A substance that is stained by an acid dye is referred to as acidophilic; it carries basic groups
which bind the acid dye. With eosin, acidophilic structures appear in various shades of pink.
Since eosin is a widely used acid dye, acidophilic substances are frequently referred to as
eosinophilic.

Neutral dyes: These are dyes that are produced from the combination of aqueous solutions of
basic and acid dyes. The precipitates that result from this combination are usually water-
insoluble but alcohol-soluble. An example is Leishman’s stain.
6.1 Staining Properties of dyes:
Just as we discussed about fixatives, dyes (stains) can be divided into two categories;
Microanatomical and cytological dyes. Microanatomical dyes generally demonstrate the
general relationship of tissues to each other. That’s e.g. the nuclei and cytoplasm are
differentiated but their structures are not emphasized. Whereas, cytological stains or dye,
demonstrate minute details of the structures in the nuclei and cytoplasm without necessarily
differentiating tissue types.

6.2 Terminologies in staining:

i) Direct staining: This is a staining process in which the tissue binds directly to the dye without
any aid.
ii) Indirect staining: this is a staining method in which the tissue binds to the dye with the help
of a mordant (a mordant is metallic substance which acts as a bridge/link between the
stain and the tissue to be stained).
iii) Accelerators: This is a substance which is incorporated into a staining solution, to increasing
the staining power of the stain.

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iv) Progressive stains: These are stains that colour tissue/cellular components in a definite order.
v) Vital stains: These are stains that stain inclusions (glycogen etc) in live cells.
vi) Metachromatic stains: In this type of stain, cellular/tissue components assume a different
colour from that of the colour of the solution they are immersed in.

6.2Characteristics of Commonly used Stains

A. Hematoxylin and Eosin (H&E)

This is one of the most widely used stains in the histology laboratory. Most of your slides are
stained with H&E. A hematoxylin-metal complex acts a as a basic dye, staining nucleic acids
in the nucleus and the cytoplasm blue, brown, or black. Eosin is an acid aniline dye which
stains the more basic proteins within cells (cytoplasm) and in extracellular spaces (collagen)
pink to red. Cartilage and mucus may stain light blue.

B. Wright's/Giemsa Stain
This and similar stains for blood and bone marrow smears are mixtures of basic (methylene blue
derivatives) and acid dyes (usually eosin). According to the number of acid and basic groups
present, cell components take up the dyes from the mixture in various proportions.

C. Metachromatic Stain

Certain basic dyes, such as toluidine blue, stain nucleic acids bluebut sulfated polysaccharides
purple (the metachromatic color). When dye molecules bound to sulfate groups are stacked
closely together, the dye experiences a color shift from blue to purple. Thus, a metachromatic
reaction often indicates the presence of numerous closely packed sulfate groups.

[Link] acid-Schiff (PAS) staining

PAS is a widely used staining technique that stains the neutral sugars of glycosaminoglycans
(this is a ground substance in most connective tissues) a pink color. Common components
stained positively with PAS include mucus, the basal lamina, and glycogen.

E. Orcein

Orcein staining is used to stain elastic fibers a dark brown-purple color. This is used, for
example, to show the elastic components in the walls of arteries, or in the matrix of elastic
cartilage.

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[Link] tetroxide

Osmium is used to stain lipids a dark black color. It is very useful for demonstrating the myelin
of myelinated nerves, or lipid droplets in the liver or steroid-secreting cells.

G. Oil Red O

Oil Red O is used to stain lipids a red-orange color in unfixed frozen sections.

CHAPTER SEVEN

DECALCIFICATION

7.0 Introduction

Decalcification
The process of decalcification is simply the process by which calcium contain elements of the
tissue are removed. Following fixation, there are techniques available to the histotechnician to
improve microtomy and staining quality.
The process of decalcification of specimens is important to prevent the difficulty to cuton a
microtome because of calcium carbonate or phosphate deposits.
The process of decalcification can be achieved either by acids or by chelating agents depending
on the circumstances.

 First, make sure the tissue has been adequately fixed and rinsedwell to prevent any
undesired reaction with the decalcifying agent.
 Dilute mineral acids (hydrochloric or nitric) or formic acid can be usedeffectively if the
end point of decalcification is monitored carefully.

Note: Nuclear and cytoplasmic details are compromised if specimens areexposed for too
long to acidic decalcifying agents, which can extractRNA and remove the purine and
pyrimidine bases from DNA.

 It is also important to wash the acid out of the tissue. If preservation of nuclearDNA is
important, or if histochemical methods for nucleic acids orenzyme activities are intended,
a chelating agent is preferred to anacid. Usually the disodium salt of EDTA is used, with
the pH adjustedto a level between 7 and 8.
 Decalcification by EDTA takes much longer thandecalcification by acids – weeks rather
than days.

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Decalcification protocol

• Make 14% EDTA solution

– Add 140 g free acid EDTA to 700 ml distilled H2O
– Place a litmus paper in the solution
– On stir plate in the fume hood, add ammonium hydroxide 30 ml at a time
until solution clears (about 90 ml total).
– Add H2O to almost 1L. Check pH and adjust with ammonium hydroxide
drop-wise up to pH 7.2 (litmus turns blue, indicating alkalinity), then
adjust final volume to 1L.

• Suspend using a waxed thread a fixed and rinsed tissue in at least 15 volumes of 14%
EDTA, and change daily or 5 times a week with mixing
– Placing tissue in a screw-top tube on its side, on a rocker works
well. Tape tubes into a rack and place the whole thing on its side
on the rocker.
• Time of decalcification varies with tissue size, species, etc.
– mouse adult long bones and vertebrae, 10-14 days
– for humans it takes about weeks
• Rinse in four changes of H2O
• for paraffin embedding, place in 30%, 50%, and 70% Ethanol for at least 30 min each
prior to submitting to core lab
• for frozen sections, blot tissue dry and freeze in OCT

Monitoring End-Point of Decalcification:

It is important for students to understand that over-decalcification can also permanently damage
a specimen. The following procedure help determine the correct end-point of decalcification.
 X-ray (the most accurate way):
This is the most satisfying method of monitoring the process of decalcification
process. Though its action can be limited by the action of a fixative like mercuric
chloride which render calcium-containing substances radio-opaque.

 Chemical testing (accurate)

This method has been one of the most used in monitoring the end-point of
decalcification, since not every facility is provided with a radiology unit.

Procedure:
1) Insert a pipette into the decalcifying solution containing the specimen.
2) withdraw approximately 5 ml of the hydrochloric acid/formic acid
decalcification solution from under the specimen and place it in a test tube.
3) Add approximately 10 ml of the ammonium hydroxice/ammonium oxalate
working solution, mix well and let stand overnight.

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4) Decalcification is complete when no precipitate is observed on two consecutive

days of testing. Repeat this test every two or three days.

 Physical testing (less accurate and potentially damage of specimen).

The physical tests include bending the specimen or inserting a pin, razor, or
scalpel directly into the tissue.
The disadvantage of inserting a pin, razor, or scalpel is the introduction of tears
and pinhole artifacts. Slightly bending the specimen is safer and less disruptive
but will not conclusively determine if all calcium salts have been removed. After
checking for rigidity, wash thoroughly prior to processing.

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CHAPTER EIGHT

BASIC TECHNIQUES

Preparation of histological sections

8.0 Introduction

In order to prepare thin sections for examination by microscopy, it is necessary to preserve the
tissues (fixation) and embed them in a supporting medium (such as paraffin wax or resins) prior
to sectioning. Sections are usually stained in order to provide contrast.

1. Fixation

In order to preserve tissues and prevent structural change or breakdown of the components of the
tissues, it is necessary to stabilize or fix the tissue. The fixative needs to preserve the tissues as
close as possible to the living state. The fixatives commonly stabilize or denature proteins. A
widely used fixative is formaldehyde, which has the advantage of being cheap and penetrates
tissues rapidly. For better fixation, it is necessary use pH buffers in the fixative.

It is important to note that, water-rich tissues are hardened by freezing and cut in the frozen state
with a freezing microtome or microtome-cryostat. This is referred to as cryosectioning
technique:

2.0 Dehydration

When tissue specimens are brought in a histology laboratory, they are in a fixative so there is
need for the tissue to be void of water, before being embedded. The solutions commonly used
are ethyl alcohol, methyl alcohol, iso-propyl alcohol. The commonly used method used is the
alcohol method.

2.1 Alcohol method: This method of tissue dehydration consists of passing the tissue through a
series of progressively more concentrated alcohol baths (or increasing grade of alcohol
concentration). The tissue is transferred from one container of alcohol to another following
allocated times, giving some few seconds to drain the alcohol from one bath to another. Ensure
that these containers containing alcohol should have lids to prevent evaporation.

Monitoring End-points of dehydration: To actually know that the tissue is water free, it is
important to keep a layer of anhydrous copper sulphate (salt) at a depth of 6mm covered with
filter paper in the container used. This salt acts as an indicator as in turns blue in the presence of
water. It’s important to note that once a blue tinge is observed, the alcohol should be discarded.

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3.0 Clearing

After the process of dehydration, it is important to understand that at this point the tissue is
water-free, and alcohol-rich. Therefore the tissue has to be cleared of alcohol by a “clearing”
agent. This process is termed “clearing” or “de-alcholisation”. Examples of clearing agents
include xylene (most commonly used), toluene, chloroform, cedar woods. A good clearing agent
should be miscible with both alcohol and paraffin wax.

4.0 Embedding/Impregnation

After the process of de-alcholisation, the tissue is alcohol-free and is subjected to a wax bath.
This molten diffuses into the tissue and replaces the “clearing agent”.

The most commonly used embedding or support medium is paraffin wax, with a melting point of
about 56°C (usually the temperature range is between 50-60 oC). Other supporting media are
gelatin, celloidin and low viscosity nitrocellulose. Histotechnicians should however understand
that some factors affect the impregnation process.

4.1 Factors that affect impregnation

a) Density of tissue: Tissues that are very dense will take a longer time for the impregnation
process to be achieved than less then tissues. The brain tissue will take longer time than the lung.

b) Size of the tissue block: its common sense for histotechnicians to know that larger block sizes
are thicker than smaller tissue blocks so therefore, will take a longer time to for molten wax to
diffuse into the tissue.

4.2 Moulds used to Embed Tissues:

i) Plastic Embedding Cassettes:

ii) Leuckart Embedding Boxes

iii) Plastic Ice-trays

4.1Frozen sections

Embedding in paraffin wax is a lengthy process and during the embedding many components
(such as lipids) are dissolved and lost. Enzymatic activities are also largely destroyed. A rapid
alternative to wax embedding is to prepare frozen sections and in a cryostat (a microtome
operated in a low temperature cabinet, usually about -30°C. Frozen sections can then be stained
or used for enzyme histochemistry, and mounted in a suitable water-soluble mountant.

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3. Microtomy

Sections of the tissue embedded in the wax block are cut on a machine, known as a microtome,
using special knives (nowadays these are disposable). Typically series or ribbons of sections are
cut at a thickness of 6-8mm. The sections are transferred to the surface of a hot waterbath (where
the sections flatten and lose any wrinkles). Sections are collected on glass microscope slides
(standard dimensions of 3 x 1 inches). In order for the sections to adhere to the slides they are
dried for up to 24 hours in a drying oven (at a temperature of about 40°C). This prevents sections
falling off the slides in the later stages of preparation.

Microtome knifes are basically classified according to their cross-section (profile) as follows:

a) Planoconcave- Hollow ground on one side

b) Wedge-shaped-plane on both sides

c) Biconcave- Hollow ground on both sides

d) Tool edge-plane on both sides with a steep cutting edge.

4. Staining

The most common staining technique is known as Hematoxylin and Eosin (or H&E) staining. In
order to stain the sections the wax needs to be removed. This is done using a wax solvent such
as xylene (xylene is considered a clearing agent since it clears away wax and water). The slide is
then hydrated using a series of descending alcohols (100%, 95%, and 70%) and then water. The
slide is then immersed in Hematoxylin stain, rinsed in running water (preferably alkaline),
followed by staining with Eosin, and rinsing in water.

4.1 H& E staining Technique

Method

1. Bring sections to distilled water

2. Stain nuclei with the alum haematoxylin

3. Rinse in running tap water

4. Differentiate with 0.3% acid alcohol (Differentiation with acid alcohol is to ascertain the
correct end-point, since the acid solution alters the colour of the tissue to red).

5. Rinse in running tap water

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6. Rinse in Scott's tap water substitute (The correct end-point is when, after blueing up, the
background is almost colourless).

7. Rinse in tap water

8. Stain with eosin 2 mins (Eosin is water-soluble acidic dye that stains the cytoplasm) .

9. Dehydrate, clear and mount.

Note: The length of time necessary to stain the tissues will depend upon fixation and the type of
alum haematoxylin employed. However, for routine tissues four minutes is required to stain the
nucleus.

Scotts Tap Water substitute is a blueing reagent designed for histology and cytology. It blues
hematoxylin gently and minimizes loss of tissue sections and cells from glass

PAS (Periodic Acid Schiff) Staining Protocol

Description: This method is used for detection of glycogen in tissues such as liver, cardiac and
skeletal muscle on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen
sections as well. The glycogen, mucin, and fungi will be stained purple and the nuclei will be
stained blue.

Fixation: 10% formalin.

Section: paraffin sections at 5 um.

Solutions and Reagents:

0.5% Periodic Acid Solution:

Periodic acid ---------------------- 0.5 g

Distilled water -------------------- 100 ml

Schiff Reagent:

Test for Schiff reagent: Pour 10 ml of 37% formalin into a watch glass. To this add a few drops
of the Schiff reagent to be tested. A good Schiff reagent will rapidly turn a red-purple color. A
deteriorating Schiff reagent will give a delayed reaction and the color produced will be a deep
blue-purple.

Mayer’s Hematoxylin Solution:

Procedure:

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1. Deparaffinize and hydrate to water.

2. Oxidize in 0.5% periodic acid solution for 5 minutes.

3. Rinse in distilled water.

4. Place in Schiff reagent for 15 minutes (Sections become light pink color during this step).

5. Wash in lukewarm tap water for 5 minutes (Immediately sections turn dark pink color).

6. Counterstain in Mayer's hematoxylin for 1 minute.

7. Wash in tap water for 5 minutes.

8. Dehydrate and coverslip using a synthetic mounting medium.

Results:

Glycogen, mucin and some basement membranes --- red/purple

Fungi ------------------------------------------------------ red/purple

Background ----------------------------------------------- blue

5. Permanent Mounting

The final stage in the preparation of tissues for microscopy is mounting. After staining the
sections are again dehydrated with ascending alcohols (95%, 100%) and xylene, prior to
covering with a mountant and a glass coverslip. Mountants need to have good optical properties.
The slide is left for at least 24 hours for the mountant to dry. The finished (permanent) slide with
its stained tissues can then be examined under the microscope.

The mountant:

 Acts as a permanent bond between slide and coverslip,

 protects cell material from air drying and shrinkage
 Acts as a seal against oxidation and fading of the stain.
 it should be colourless and transparent
 it should be able to completely permeate and fill tissue interstices
 it should have no adverse effect on tissue components
 it should be resistant to contamination (particularly microorganism growth)
 it should protect the section from physical damage and chemical activity
(oxidation and changes in pH)
 it should be completely miscible with dehydrant or clearing agent

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Types of mountants:

Mounting media are either hydrophobic or hydrophilic.

a) Hydrophobic mountants (resinous): Have a higher refractive index (RI)

i) Canada Balsam

This is an oleoresin obtained from the bark of the fir Abiesbalsamea (of the family Pinaceae),
native to North America. The dried resin is freely soluble in xylene and other organic solvents,
Canada balsam has a number of disadvantages: it yellows with age; is very slow to harden and;
as it becomes increasingly acidic over time, cationic dyes are poorly preserved and the Prussian
blue product of Perls' reaction is bleached.

ii) DPX (Distrene, Plasticiser, Xylene)

One of the most commonly used mountants; DPX is a colourless, neutral medium in which most
standard stains are well preserved. It is prepared by dissolving the common plastic, polystyrene,
in a suitable hydrocarbon solvent (usually xylene). A major disadvantage of polystyrene media,
however is that they set quickly and in doing so often retract from the edge of the coverslip. This
can be prevented by adding a plasticiser which is thought to resist the effect by forming a mesh
with the polymerised plastic.

Note: Resin-Embedded Tissue: Sections of tissue embedded in plastic compounds (such as

epoxy resins) can be successfully mounted in liquid resin of the same type. Sections should be
completely dry before applying mountant which is best set using the same conditions prescribed
for tissue blocks.

b) Hydrophilic (aqueous) mountants: Have a lower RI, water-miscible.

i) GLYCEROL

Glycerol is also a useful temporary mountant but with a higher RI (1.460) and longer drying time
than water. Ringing the coverslip with a hydrophobic seal will extend the life of mounted
sections, although cationic dyes will diffuse into the medium over time. Phosphate buffered
glycerol (RI 1.47) is commonly used to mount sections for immunofluorescence14 and glycerol
may be added to other agents to retard drying and cracking.

iii) GLYCERINE (GLYCEROL) JELLY

This commonly utilised aqueous mountant is a mixture of glycerol and gelatine and has a RI
of 1.47. It should set quite hard but for long term preservation sections are best ringed and
sealed. Various formulations are in use.

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SECTION II

Cytologic Techniques

1.0 Introduction
Cytology simply refers to the study of the cell structure and function. This is further expanded
into exfoliative cytology which seeks to explore the microscopic examination of cells that are
spontaneously shed or exfoliated from the epithelial surfaces of the body. Students should recall
that it was earlier mentioned that regeneration of epithelial cells occur around the basal laminar,
and the pushes upwards to replace old ones which slough-off. Cancerous cells will more readily
exfoliate than normal cells. Exfoliated cells can be found in epithelial membrane or cavities of
the vagina, buccal cavity, and pleural fluid…It is also important to draw a fine line between
cytology and histology. In histology, the histotechnician tries to evaluate malignancy by
examining aggregated cells (tissue), whereas here the professional is interested in individual cells
or a small number of cells. The Cytotechnician/Histotechnician is interested in understanding the
cytoplasm-to-nucleus ratio and other aspect of cell division and maturation.

Histology of the cervix

An understanding of the histology of the cervix is critical to the use of effective cytologic
screening, and biopsy results in the management and treatment of cervical neoplasia. The stroma
of the cervix, which accounts for most of its mass and shape, is composed of dense,
fibromuscular tissue made up of collagenous connective tissue (smooth muscle and elastic tissue)
and ground substance (mucopolysaccharide).
The cervix is covered by both columnar and stratified non-keratinising squamous epithelia. The
squamocolumnar junction, where these two meet, is the most important cytologic and
colposcopic landmark, as this is where over 90% of lower genital tract neoplasia arises. This
junction is presumed, but not proven, to be the embryologic junction of the Müllerian and
urogenital sinus epithelia.

Transformation Zone
An understanding of the cervical transformation zone (TZ) is essential to the identification and
management of cervical intraepithelial neoplasia. It lies between the “original” and “new”
squamocolumnar junctions. The SCJ discussed above is the visible border between the squamous
and columnar epithelia of the cervix and represents the new squamocolumnar junction.

Some of these nucleus-associated abnormalities include:

 Decrease cytoplasm: nucleus ratio because of an enlargement of the nucleus
without an overall increase in cell size
 Nuclear abnormality regarding the outline/ nuclear variation in size and shape
 Hyperchromasia associated with increased amount of DNA
 Multinucleation as a result of abnormal cell division
 Uneven distribution and variation in size of chromatin particles.

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These abnormalities are associated to neoplasia and dysplasia

a) Neoplasia: This is the uncontrolled disorderly proliferation of cells, resulting in a benign
or malignant tumour.
b) Dysplasia: This is a reversible change which often precedes malignancy. This is seen as
disorderly maturation and spatial arrangement of cells marked variability in nuclear size
and shape and increased often abnormal mitosis.

Collection of cytological specimens

Collection in the cytology laboratory is very important, because if specimens are not well
collected then the right diagnosis will not be issued. Different instruments are used for the
collection of specimens at specific sites.
a) Vaginal smears: Materials for cytological analysis are collected from the posterior
vaginal fornix (upper third) with an aspirating bulb or pipette (it’s a stout-walled, slightly
curved glass pipette fitted with a rubber bulb) or gently scrapping the lateral wall with a
plastic or wooden spatula (Ayre spatula is designed with one end being shaped to
penetrate the cervix).
b) Cervical smears (Endocervical):The cervix is located at the top of the vagina. It is the
opening to the uterus and is composed of dense connective tissue. It has very little
smooth muscle in it, compared to the rest of the uterus, which is almost entirely smooth
[Link] for cytological analysis can be collected with the help of a speculum.
This appliance is inserted into the vagina and allows for the uterine cervix to be observed
directly. A cotton-wool swab, a spatula or a cyto-brush is inserted into it, and swabbed
gently.
c) Sputum smears: early morning specimens are collected from productive deep cough.
The sputum is then macroscopically examined for particles or blood. Then fixing follows.

1.2 Fixing of cytological specimens

Cytotechnician /Histotechnicians sometimes perform special stains on cytology smears, blood
films and cytological-preparations (cytopreps) from other departments within thelaboratory.
Increasingly, the commonly received cytoprep is that of the thin preparation on clean slides.
These smears are wet-fixed in 95% ethanol immediatelyafter preparation to preserve the fine
structure of the chromatin andhelp in the evaluation of nuclear changes.
Sprays: There are sprays intended for laboratory use and hair spray is also used. If the latter is
used, it is advisable to buy economic brands because they have more alcohol and less shellac.
.

1.3 Staining cytological specimens

As earlier explained in histology, staining plays a crucial role in cytology in deciphering
pathologies: Understanding both the physical (size of dye and porosity of tissue/cellular
component) and chemical factors.
The May-Grunwald orthe Giemsa stain is routinely evaluated, and the more
complicatedPapanicolaoumethodis widely used, especially on samplestaken from the vagina

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and cervix. Air drying is avoided with smears for cytological detection of neoplasia because it
changes the appearances of the cells.
Slides bearing blood or bone marrowsmears, on the other hand, are usually air-dried. Marrow
smearsare stained in parallel to sections of the bone marrow core biopsy
1.3.1 Papanicolaou staining
Papanicolaou staining is the quintessential technique to general cytology just as hematoxylin-
eosin to the histological method. Below it describes the complete original technique together
with practical and everyday variations and other useful tips that it hopesstudent will find useful.
Adequate material collection, correct and rapid fixation and good staining process are required to
achieve a good cytological preparation.
What is a Pap smear?
A Pap smear (also known as the Pap test) is a medical procedure in which a sample of cells from
a woman's cervix (the end of the uterus that extends into the vagina) is collected and spread
(smeared) on a microscope slide. The cells are examined under a microscope in order to look for
pre-malignant (before-cancer) or malignant (cancer) changes.
In the vast majority of cases, a Pap test does identify minor cellular abnormalities before they
have had a chance to become malignant and at a point when the condition is most easily
treatable.
Who should have a Pap smear?
 Any lady who has had a sexual intercourse
 Any woman who is pregnant.
 Women having difficulties in conceiving
 Women above the age whether sexually active or not.

1.4 Papanicolaou staining Technique

For Papanicolaou staining, preparations need to be previously fixed in 96º alcohol. The material
spread on the slide should be fixed immediately before drying begins. Ideally, it should be
immersed in 96º alcohol for no less than 10 minutes.
The smear should be uniformly sprayed for some seconds at a distance of not less than 15-20
cm. Preparations dry quite rapidly but if they are paper wrapped before they dry, paper cellulose
fibers usually adhere to them.
Preparations fixed in this way may be preserved for up to two weeks before staining, making it
possible to send them far away if there are no laboratories available nearby.
Smears may be re-hydrated provided there was no prior fixation.
The PAP stain is designed to meet 3 staining objectives:
1. Good nuclear detail,
2. Differential counterstaining,
3. Cytoplasmic transparency.

Cytoplasmic transparency is a function of high ethanolconcentration of the stain. This is

important in orderto view multilayered cell aggregates.
The vital components of the PAP stain are:
1. Harris's haematoxylin as a nuclear stain,
2. Orange G,

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3. Eosin Alcohol 50 (EA50)

Re-hydration:
Procedure: Put the sample in a solution of distilled water and glycerin in equal parts (a/a) for 3 to
5 minutes.
Example: 50 ml of distilled water and 50 ml of glycerin (3-5 minutes).
Following that, drain the sample and fix to in 96º alcohol or fixing spray.
Water hydrates cells and glycerin restores elasticity to them.
If you do not have those elements.
Immerse the sample for hydration directly in running tap water for 3-5 minutes and fix with 96º
alcohol or spray. Afterwards, stain as usual.

The Papanicolaou staining technique is very versatile and has undergone numberless variations
resulting in good quality products suitable for the meticulous microscopic observation required
by cytology.
It is a very refined, scientifically-proved staining method which requires a high number of steps
in its original formulation and yields a product with absolute subtlety of colors and transparency.
Original Papanicolaou staining method:
1. 96º ethyl alcohol for15 sec
2. 70º ethyl alcohol for 15 sec
3.50º ethyl alcohol 15 seconds
[Link] water 15 seconds
5. Harris hematoxylin 6 min (hematoxylin is a basic dye that stains acidic nuclei of cells
revealing chromatinic patterns).
6. Distilled water 10 dips
7. Hydrochloric acid 0.5% solution, 1-2 quick dips
8. Distilled water 15 sec.
9. Ammonia 1.5% solution in 70º alcohol: The smear turns to blue (This subjects the
haematoxylin to alkaline conditions and changes its colour from red to blue.
10. 50º ethyl alcohol 15 sec
11. 70º ethyl alcohol 15 seconds
12.96º ethyl alcohol 15 sec
13. OG-6 (Orange G) 2 min: Orange G solution is used to stain cytoplasms. It is an alcoholic
solutions and alcohol makes cytoplasms more transparent.
14. 96º ethyl alcohol 10 dips
15. 96º ethyl alcohol 10 dips
16. EA 50( Eosin Alcohol) for 3 minutes: EA 50 is a mixture of eosin, light green and
Bismarck brown that stains the cytoplasmand enhances its transparency)
17. 96º ethyl alcohol (10 dips)
18. 100º ethyl alcohol (10 dips)
19. Xylol (or xylene) (10 dips): This is a clearing agent.
20. Mount: with Canada balsam and cover with glass cover slips large enough to cover the entire
sample.
Cover Slips: Cervical-vaginal samples should not be smaller than 24 x 50 mm. smaller sizes
indicate insufficient samples.

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Recommendation: It is very important to respect the hematoxylin time. Nucleus over-staining

results in hyperchromasia and may lead to misinterpretation of nuclear abnormalities and
overdiagnosis.
On the other hand, staining time with OG-6 and EA is less demanding because they act by
saturation and excess time does not cause consequences as dramatic as hematoxylin overstaining.
The OG-6 (Orange G) stains keratin in the smear, while the EA (Light Green SF or Fast Green
FCF) is respnsible for the green cytoplasmic staining of squamous cells before they keratinize. It
is very difficult to interpret cytologic preparations when this stain is inadequately balanced, and
it is the job of the cytotechnologist to see this done right.
Rapid Papanicolaou staining (modified method)
1. 96º alcohol
2. Running tap water 10 passes
3. Hematoxylin 40 sec
4. Wash in running tap water until it gets transparent, without dye traces
5. 96º alcohol 10 passes
[Link]-6 40 sec
7.96º alcohol 10 passes
[Link] 40 sec
9.96º alcohol 10 passes
10.100º alcohol 10 passes
11.100º alcohol
12. Xylene 10 passes
13. Mount

Whether in a histology or cytology laboratory, technicians should always institute SOPs to avoid
faulty staining results:
Causes of inconsistent/faulty staining
1. Varying thickness of material on slide
2. Type of fixative used
3. Inadequate filtering of stain solutions
4. Age of staining solution
5. Degree of usage of staining solutions
6. Use of chlorinated tap water
7. pH of water can effect nuclear staining
8. Temperature of water
9. Insufficient rinsing after acid
10. Air drying of slides between solutions

PAP SMEAR
1. WHAT IS A PAP SMEAR?
2. WHAT MATERIAL DO YOU USE TO COLLECT A PAP SMEAR?
3. WHAT ARE THE CONDITIONS FOR COLLECTING A PAP SMEAR?
4. HOW MANY SLIDES ARE COLLECTED FOR A PAP SMEAR?
5. WHAT PARTS OF THE FEMALE GENITAL TRACT ARE SMEARED?
6. WHAT ARE THE METHODS USED IN FIXING A PAP SMEAR?
7. WHAT ARE THE REAGENTS USED TO STAIN A PAP SMEAR?

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8. WHAT OTHER STAINING TECHNIQUES CAN YOU USE FOR CERVICAL SMEARS?
9. HOW OFTEN DOES A WOMAN DO PAP SMEARS?
10. AT WHAT AGE DOES SHE START HAVING PAP SMEARS?

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