Chapter One:

Introduction to Histopathology:
Learning Outcomes:
At the end of the study the learner should be able to:
 Define terms related to histopathology
 Describe the importance of histopathology
 Describe the historical background of pathology
 Outline the development of cellular pathology

Historical background of pathology:

Histology is the microscopic study of normal body tissues while the study of diseased
tissues is referred to as Histopathology. It has close relationship with other disciplines
particularly Cell Biology, Pathology, Biochemistry, Cytology and Immunology.
Histology is derived from a Greek word `Histos’ meaning tissues and `logia’ meaning
study of or knowledge. It is necessary for Histotechnician to understand the nature of
disease in order to apply histological methods to the required standards.
The word ‘pathology’ is derived from two Greek words ‘pathos means suffering and
logos’ meaning study. Pathology is thus the scientific study of structure and function
of the body in disease. It deals with causes, effects, mechanism and nature of disease.
The knowledge and understanding of pathology is essential for all would be doctors as
well as general practitioners and specialists since unless they know the causes and
mechanisms of diseases and understand the language of pathologists in the form of
laboratory reports. They would not be able to institute treatment or suggest preventive
measures to the patient.
The earliest concepts of disease were the religious beliefs that affliction or disease was
the outcome of curse or evil spirits. To ward them off, Priests through prayers and
sacrifices used to invoke supernatural power to please the gods.
The period of ancient religious believes was followed by the philosophical approach
and rational thinking to disease by methods of observations. This happened at the time
when great Greek philosophers like Socrates, Plato and Aristotle introduced
philosophical concepts to all natural phenomenons.
The real practice of medicine began with Hippocrates (460-377 BC), the Greek clinical
genius of all times and regarded as the father of medicine. He stressed the study of

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patients’ symptoms and described methods of diagnosis. According to him,
disturbances in equilibrium of the body resulted in illness.
Hippocrates introduced ethical concepts in the practice of medicine and is revered by
medical practitioners by taking ‘Hippocratic oath’ at the time of entering into the
profession.
Cornelius Celsius (53 BC- 7AD) describe four cardinal signs of inflammation – Rubor
(redness), Tumour (swelling) Calor (heat) and dolour (pain).
Various scientists (7AD– 1800AD) studied various factors related to disease among
them Von Leeuwenhoek (1632 – 1723) who invented the first microscope and Marcelo
Malpigi who described the malpigian layer of the skin and the lymphoid tissue.
Development of Cellular Pathology:
Upto the middle of 19th century correlation of clinical manifestations of disease with
pathological findings at autopsy became the major method of study of disease.
Pathology started developing as a diagnostic discipline in the later part of the 19 th
century with the evolution of cellular pathology.
This was closely linked to technology advancement in machinery manufacture for
cutting thin sections of tissue, improvement in microscope, and development of
chemical industry and dyes for staining.
Among the notable scientists who played a great role in the development of cellular
pathology include;
1. Paul Erlich (1854 – 1915). He described Erlich’s test for urobilinogen using
Erlich’s aldehyde reagent, staining techniques of cells and bacteria and laid
foundation for Haematology.
2. Christian Gram (1853-1938), developed bacteriological staining by crystal
violet.
3. D.L.Romanowsky (1861-1921), developed stain for peripheral blood using
eosin and Methylene blue derivatives.
4. Robert Koch (1843-1910), developed techniques of fixation and staining for
identification of bacteria, discovered Tubercle bacilli in 1882 and Vibrio
cholera organism in 1883.
5. May-Grunwald, developed May- Grunwald stain in 1902 and Giemsa stain in
1914. He applied them for classification of blood cells and bone marrow.
6. Sir William Leishman (1865-1926), developed Leishman stain for blood films
in 1914 and observed Leishmania Donovani bodies (LD bodies) in
leishmaniasis infection.
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 7. Robert Feulgen (1884-1955). He described Feulgen reaction for DNA staining
and laid the foundations of Cytochemistry and Histochemistry.
8. Karl Landsteiner (1863-1943), described the existence of blood groups in
1901.
9. George N. Papanicolaou (1883-1962), developed exfoliative cytology for early
detection of cancer and is usually referred to as the father of cytology.
The development has made it possible to study diseases at molecular level, and provide
an evidence-based and objective diagnosis and therapy.
The major impact of advances in molecular biology are in the fields of diagnosis and
treatment of genetic disorders, immunology and in cancer.
It is applied in the study of plant and animal tissues. Histological study provides the
following information:
1. Knowledge of cellular structures of different parts of the body thus providing
understanding of the various body organs.
2. Changes due to inflammatory process.
3. Recognition of cancer cases.
4. Diagnosis of Parasitic, Bacterial, fungal and Viral infections.
In human histology specimens for histological examination are obtained during post-
mortem or surgical procedures. Post-mortem specimens are referred to as autopsies
while those removed during surgery are known as biopsies.
Tissues undergo changes immediately after removal from the body or after death. To
get a clear picture of the status of the tissue as it was in the body it must be examined
immediately. The tissue can also be preserved using special techniques if it cannot be
examined as soon as possible.
There are several techniques (methods) that can be used to prepare tissues for
microscopic examination. Some of these methods have a wide application and produce
some interesting results such that one should be acquainted with the principles
underling them. The methods involved are:
(a) Direct examination of cells and tissues: This is done using the various
microscopes available .They include, Ordinary light, Polarised light,
interference and dark ground microscopes. The examination is on either fresh
or preserved specimens.
(b) Quantitative and qualitative methods: These are methods that are used to
determine the composition of cells and tissues; they could be physical or
chemical methods. Physical methods include the use of Fluorescence
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 microscopy, scanning electron probe (Micro-analysis) and Autoradiography.
The chemical methods are varied; they use various chemicals to determine the
presence of important organic and other molecules within the cells and tissue
(This is referred to as Histochemistry).
Microscopic examination of tissues
Tissues are sliced into thin pieces or sections for the examination; they are either
examined when fresh or after preservation (fixation).
The selected technique for preparation is governed by the following factors:
(a) The type of microscope to be used
(b) Structures or inclusions of tissues to be studied
(c) The amount and nature of tissue
(d) Urgency of the examination
(e) Whether tissue is fresh or fixed
Types of tissue preparation
Fresh tissues can be examined by use of the following preparations:
Teased preparation;
A tissue is put in isotonic solution then dissected with mounting needles to obtain thin
sections. The slices are then transferred onto a glass slide and mounted with a cover
glass then examined as a wet preparation. Stains such as Methylene blue can be used to
provide a clear contrast of the structures under study.
Teased preparations are useful during the study of cells undergoing cell division
(Mitosis).
Squashed preparation;
A thin slice of tissue not exceeding 1mm thick is put on a glass slide and forcefully
covered with a cover glass. Staining if necessary is done by capillary attraction through
the sides of the cover glass.
This type of preparation is useful during the study of cellular contents like
Mitochondria.
Smears;
Smears are prepared in several ways depending on the nature of the material to be
examined. They are examined when fresh or fixed either stained or unstained.
They can be made by spreading the selected portion of the material over the surface of
a glass slide using a platinum wire loop.
An apposition smear can also be made with the aid of a second glass slide. The
material is put on the slide and is spread by use of another slide.
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The fresh cut surface of the piece of tissue is made to touch surface of the slide, this
leaves some of the contents of the tissue on the slide. This type of smear is known as
an impression or touch smear.
Frozen sections;
This involves freezing of fresh tissues and thin sections (5-10 microns) are cut using
special instruments called microtomes, an example of such an instrument is the
freezing microtome. The cooling is done by use of compressed carbon dioxide, liquid
Nitrogen or electro-thermal coupling units.
The cut section is transferred onto a slide or into a dish containing isotonic saline
where it can later be put on the slide and stained.
Some cellular tissue structures are soluble in processing solutions, others are distorted
by heat. Such structures include; enzymes, fats, some proteins and antigens. Frozen
sections are also necessary when dealing with specimens that require urgent
investigations.
Preserved tissues:
Tissues when removed from the body as either biopsies or autopsies undergo changes
known as post mortem changes. These changes can be retarded and prevented by
preservation of the tissue using low temperature or by use of chemical preservatives
called fixatives. The post mortem changes that are encountered are Putrefaction and
Autolysis.
Putrefaction:
This is the breakdown of tissue after death or removal of tissue/cells by the action of
bacteria. The bacteria that were previously commensals like those in the alimentary
canal or may have been causative agents of disease and even from the environment are
responsible for putrefaction.
Autolysis:
It is the destruction of tissue by its own enzymes. Immediately the cell dies, the
lysosome raptures, releasing lytic enzymes. This digest the surrounding tissues, these
enzymes belong to cathepsin group.
Signs of autolysis
(a) The nucleus first condenses in the process called Pyknosis. It then fragments
(Karyorrhesis) and finally disappears in the process called Karyolysis.
(b) The cytoplasm swells, becomes granular and eventually becomes a
homogenous mass with the loss of normal staining power.

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 (c) The glycogen within the cytoplasm diminishes or diffuses out of the cell
leaving an empty space
(d) The epithelial tissue sheds off and split away from the basement membrane.
Any person responsible for study of histological material must be conversant with
normal and abnormal structure of cells and tissues in order to apply the proper
techniques for good results during microscopic examination. It is for this reason that a
brief description has been included in this manual,

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 Chapter Two:

Normal Human Cell:

Learning outcomes:
At the end of instructions the learner should be able to;
 Draw and label the human cell.
 Describe the basic parts of the cell.
 Illustrate mitosis diagrammatically.
 Differentiate between mitosis and meiosis.
 Draw and label the structure of DNA molecule.

Introduction
Human body unlike unicellular amoeba, is quite complex and is made up of 70,000
billion cells which comprise different tissues and organs, each of which is assigned
predetermined specific functions. Cells are the smallest functional units of the body,
they are grouped together to form tissues. Different types of cells of the body posses
features which distinguish one type from another. However, most mammalian cells
have an overall common structure and function.
Cell structure:
Under normal conditions, cells are dynamic structures existing in fluid environment. A
cell is enclosed by cell membrane that extends internally to enclose nucleus and
various subcellular organelles suspended in Cystosol (cytoplasm). Organelles are small
structures with highly specialized functions, many of which are contained within a
membrane.
a) Cell Membrane (plasma membrane)
The plasma membrane consists of two layers of phospholipids (fatty substances) with
some protein molecules embedded in them. Biochemically the cell membrane is
composed of complex mixture of phospholipids, glycolipids, cholesterol, proteins and
carbohydrates. These layers are in a gel-like arrangement and are in a constant state of
flux. The outer surface of some types of cells shows a coat of mucopolysaccharides
forming a fuzzy layer called glycolcalyx. Proteins and glycoproteins of the cell
membrane may act as antigens like blood group antigens or may form receptors for
viruses, bacterial products, hormones, immunoglobulins and many enzymes.

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The cell receptors are probably related to the microtubules and microfilaments of the
underlying cytoplasm. The microtubules connect one receptor with the next. The
microfilaments are contractile structures so that the receptor may move within the cell
membrane. Bundle of microfilaments along with cytoplasm and protein of cell
membrane may form projections on the surface of the cell called microvilli. Microvilli
are numerous on the surface of absorptive and secretory cells like small intestinal
mucosa.
The cell membrane performs the following function;
i) Selective permeability that includes diffusion, membrane pump (sodium
pump) and pinocytosis (cell drinking).
ii) Membrane antigens such as blood group antigens and transplantation
antigens thus giving the cell its immunological identity.
iii) Cell receptors for cell recognition and communication. Act as specific
receptors for hormones and other chemical messengers.

Human cell

b) Nucleus

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The nucleus consists of an outer nuclear membrane enclosing nuclear chromatin and
nucleoli. Every cell in the body has a nucleus, with the exception of mature
erythrocytes (red blood cells). The nucleus is contained within a membrane similar to
plasma membrane.
Nuclear membrane:
The nuclear membrane is the outer most envelop consisting of 2 layers. The outer layer
of the nuclear membrane is studded with ribosomes and is continuous with
endoplasmic reticulum.
The two layers of nuclear membrane at some places are fused together forming circular
nuclear pores which are about 50 nm in diameter.
The nuclear membrane has tiny pores through which some substances can pass
between it and the cytoplasm.

Nuclear chromatin:
The main substance of the nucleus is comprised of the nuclear chromatin which is in
the form of shorter pieces of thread like structures called chromosomes of which there
are 23 pairs (46 chromosomes). 22 pairs (44 chromosomes) are autosomes and one pair
of sex chromosomes, either XX (female) or XY (male). Each chromosome is
composed of two Chromatids connected at the centromere to form ‘X’ configuration
having variation in location of the centromere.
Chromosomes are composed of 3 components, each with distinctive function. These
are deoxyribonucleic acid (DNA) comprising about 20%, ribonucleic acid (RNA)
about 10% and the remaining 70% consists of nuclear proteins that include a number
of basic proteins (histones), neutral proteins and acid proteins.

Chromosome: It is a very long DNA molecule and associated proteins that carry
portions of the hereditary information of an organism.

a. Structure of a chromosome: A chromosome is formed from a single DNA

molecule that contains many genes. A chromosomal DNA molecule contains

three specific nucleotide sequences which are required for replication: a DNA

replication origin; a centromere to attach the DNA to the mitotic spindle.; a

telomere located at each end of the linear chromosome.

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 The DNA molecule is highly condensed. The human DNA helix occupies too much

space in the cell. Small proteins are responsible for packing the DNA into units

called nucleosomes.

b. Stained chromosomes: Chromosomes are stained with A-T (G bands) and G-C

(R-bands) base pair specific dyes. When they are stained, the mitotic

chromosomes have a banded structure that unambiguously identifies each

chromosome of a karyotype. Each band contains millions of DNA nucleotide

pairs which do not correspond to any functional structure.

c. Karyotype of a male: The human haploid genome contains 3,000,000,000 DNA

nucleotide pairs, divided among twenty two (22) pairs of autosomes and one
pair of sex chromosomes.

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DNA of the cell is largely contained in the nucleus. The only other place of the cell

that contains the DNA is the mitochondria. DNA carries the genetic information that is

passed via RNA into the cytoplasm for manufacture of the proteins of similar

composition. During cell division, one half of DNA molecule aces as a template for the

manufacture of the other half by the enzyme, DNA polymerase, so that the genetic

characteristics are transmitted to the next progeny of cells.

The DNA molecule consists of two complimentary polypeptide chains forming a

double helical strand which is wound spirally around an axis composed of pentose
sugar -phosphoric acid chains. The molecule is spirally twisted in a ladder-like pattern
the steps of which are composed of 4 nucleotide bases; two purines (adenine and
guanine) and two pyrimidines (cytosine and thymine). Adenine (A) pairs specifically
with Thymine (T), while, Guanine (G) pairs with Cytosine (C). The sequence of these
nucleotides pairs in the chain, of which there are thousands, determines the
information contained in the DNA molecule or constitutes the genetic code.

Nucleolus;
The nucleus may contain one or more rounded bodies called nucleoli. These are the
site for synthesis of ribosomal RNA. Nucleolus is composed of granules and fibrils
representing newly synthesized ribosomal RNA.

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c) Cytosol and organelles:
The cytosol or cytoplasm is the gel-like ground substance in which organelles of the
cells are suspended. These organelles are sites of major enzymatic activities of the cell
which are possibly mediated by enzymes in the cytoplasm. The major are the
cytoskeleton, mitochondria, ribosomes, `endoplasmic reticulum, golgi apparatus,
lysosomes and micro-bodies.
1. Cytoskeleton;
Microfilaments, intermediate filaments and microtubules are responsible for
maintaining cellular form and movement and are collectively referred to as
cytoskeleton
i) Microfilaments are long filamentous structures. They are composed of contractile
proteins, actin and myosin, and diverse materials like ribonucleic protein fibres.
Bundles of microfilaments are especially prominent close to the plasma membrane and
form terminal web. Extensions of these bundles of microtubules along with part of
plasma membrane on the surface of the cell form microvilli which increase the
absorptive surface of the cells.
ii) Intermediate filaments are cytoplasmic structures found in a number of cell types.
They are composed of proteins. There are 5 principal types of intermediate filaments.
a) Cytokeratin (found in epithelial cells)

b) Desmin (found I skeleton, smooth and cardiac muscle)

c) Vimentin (found in cells of mesynchemal origin)

d) Glial fibrillary acidic protein (present in astrocytes and ependymal cells)

e) Neurfilaments (seen in neurons of central and peripheral nervous system).

iii) Microtubules are long tubular structures. They are composed of protein, tubulin,

cilia and flagella which project from the surface of cell. Cilia and flagella are active in

locomotion of cells.

2. Mitochondria;

These are oval or sausage-shaped structures in the cytoplasm, sometimes described as

the power house of the cell. They are involved in cellular respiration, the process by

which chemical energy is made available in the cell. Chemically and structurally,

membranes of the mitochondria are similar to cell membrane. The chemical energy is
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in the form of adenosine triphosphate (ATP). ATP is a high energy unstable compound

formed from the catabolism of carbohydrates, fats and if these are in short supply.

Proteins, Synthesis of ATP is most efficient in the final stages of cellular respiration.

This is called oxidative phosphorylation, requiring the presence of oxygen. Aside from

their role in metabolism, mitochondria possess DNA and Ribosomes, but no histones,

and may have a role in synthesis of membrane bound proteins of mitochondria.

3. Ribosomes;

Ribosomes are spherical particles which contain 80-85% of the cells RNA. They may

be present in the cystosol as free unattached form, or in bound form when attached to

the membrane of endoplasmic reticulum. They may lie as monomeric units or as

polyribosomes, when many monomeric ribosomes are attached to a linear molecule of

messenger RNA. Ribosomes are the proteins synthesizing units from amino acids.

When present in free units or in small clusters in the cytoplasm, the ribosomes make

proteins for use within the cell.

4. Endoplasmic reticulum;

Endoplasmic reticulum is composed of vesicles and intercommunicating canals whose

functions is the manufacture of proteins. It is composed of unit membrane which is

continuous with both nuclear membrane and golgi apparatus and possibly with the cell

membrane. Morphologically, there are two forms of endoplasmic reticulum, rough or

granular and smooth or agranular.

Rough endoplasmic reticulum is so called because its outer surface is rough or granular

due to attached ribosomes on it. Rough endoplasmic reticulum is well developed in

cells active in protein synthesis such as Russel bodies of plasma cells and Nissl

granules of nerve cells.

Smooth endoplasmic reticulum is devoid of ribosomes on it surface. They contain

many enzymes that are involved in synthesis of lipids and steroids hormones and are

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also associated with detoxification of some drugs and metabolism of cholesterol,

carbohydrates and partake in muscle contraction.

5. Golgi apparatus (Complex);

The golgi apparatus is generally located next to the nucleus. Morphologically, it

appears as vesicles, sacs or lamella composed of unit membrane and is continuous with

endoplasmic reticulum. The golgi apparatus is particularly well developed in exocrine

glandular cells. Its main functions are synthesis of carbohydrates and complex proteins

and packaging of proteins synthesized in the rough endoplasmic membrane into

vesicles.

6. Lysosomes;

Lysosomes are rounded to oval or spherical membrane-bound bodies/organelles

containing powerful lysosomal digestive (hydrolytic) enzymes. They are involved in
breaking fragments of organelles and large molecules such as RNA, DNA,
carbohydrates and proteins inside the cell into smaller particles that are then extruded
from the cell as waste material. Lysosomes in white blood cells contain enzymes that
digest foreign material such as microbes.
7. Centriole or Centrosome;
They are seen two small structures composed of dense fibril. They perform the
function of cilia and flagella and form the spindle during mitosis.

Cell cycle:
Multiplication of the somatic and germ cells is the most complex of all the cell
functions. It is controlled by genes which encode for release of specific protein
molecules that promote or inhibit the process of mitosis at different steps. Mitosis
promoting protein molecules are cyclins A, B and E. these cyclins activate cyclin-
dependent kinases (CDKs) which act in conjunction with cyclins. After the mitosis is
complete, cyclins and CDKs are degraded and the residues of used molecules are taken
up by cytoplasmic caretaker proteins, ubuquitin, to the peroxisome for further
degradation.
Mitosis
Mitosis divides the chromosomes in a cell nucleus.Mitosis is the process by which a
eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in

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two nuclei. It is generally followed immediately by Cytokinesis, which divides the
nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly
equal shares of these cellular components. Mitosis and Cytokinesis together define the
mitotic (M) phase of the cell cycle - the division of the mother cell into two daughter
cells, genetically identical to each other and to their parent cell. This accounts for
approximately 10% of the cell cycle.

Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different
species. Prokaryotic cells, which lack a nucleus, divide by a process called binary
fission.

The process of mitosis is complex and highly regulated. The sequence of events is
divided into phases, corresponding to the completion of one set of activities and the
start of the next. These stages are prophase, metaphase, anaphase and telophase.
During the process of mitosis the pairs of chromosomes condense and attach to fibres
that pull the sister Chromatids to opposite sides of the cell. The cell then divides in
Cytokinesis, to produce two identical daughter cells. The period between mitosis is
called Interphase

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Phases of cell cycle and mitosis

Interphase

The mitotic phase is a relatively short period of the cell cycle. It alternates with the

much longer Interphase, where the cell prepares itself for cell division. Interphase is

therefore not part of mitosis. Interphase is divided into three phases, G 1 (first gap or

pre-mitotic gap), S (synthesis), and G2 (second gap). During all three phases, the cell

grows by producing proteins and cytoplasmic organelles. However, chromosomes are

replicated only during the S phase. Thus, a cell grows (G1), continues to grow as it

duplicates its chromosomes (S), grows more and prepares for mitosis (G 2), and finally

divides (M) before restarting the cycle.

G1 phase– This is the stage when messenger RNAs for the proteins and the proteins

themselves required for DNA synthesis (e.g. DNA polymerase) are synthesis. The

process is under control of cyclin E and CDKs.

S phase – Involves replication of nuclear DNA. Cyclin A and CDKs control it.

G2 phase – Is the short gap phase in which correctness of DNA synthesized is

assessed. This stage is promoted by cyclin B and CDKs.

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M phase – is the stage in which process of mitosis to form two daughter cells is

completed. This occurs in four sequential phases/stages; prophase, metaphase,

anaphase and telophase.

G0 – The daughter cells may continue to remain in the cell cycle and divide further, or

may go out of the cell cycle into resting phase called G0 phase.

Prophase

The two round objects above the nucleus are the centrosomes; they divide and move

towards the opposite poles. Chromatin in the nucleus begins to condense and becomes

visible in the light microscope as chromosomes. Each chromosome divides into two

Chromatids which are held together by centromeres.The nucleolus disappears.

Centrioles begin moving to opposite ends of the cell and fibres extend from the

centromeres. Some fibres cross the cell to form the mitotic spindle.

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Metaphase

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.

Spindle fibres align the chromosomes along the middle of the cell nucleus and are held
in place by microtubules attached to the mitotic spindle. This line is referred to as the
metaphase plate (equatorial 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

Kinetochore microtubules (spindles) shorten as they move to the opposite poles of the

cells. The centromeres divide and the paired chromosomes separate and move to

opposite sides of the cell. Sister Chromatids separate and move towards the

corresponding poles.

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Telophase

The decondensing chromosomes are surrounded by nuclear membranes. Note

Cytokinesis has already begun, the pinching is known as the cleavage furrow.

Chromatids (Daughter chromosomes) 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 fibres disperse and disappear.

The cell nuclear envelope reappears. The cytoplasm divides, the cell membrane
pinches inward ultimately producing two daughter cells (phase: Cytokinesis).

Meiosis:

In meiosis, the phases are, analogous to mitosis: prophase II, metaphase II, anaphase
II, and telophase II (see figure below). As shown in the figure below, meiosis II begins
with two haploid (n = 2) cells and ends with four haploid (n = 2) cells. Notice that
these four meiocytes are genetically different from one another. In humans (2n = 46),
who have 23 pairs of chromosomes, the number of chromosomes remains unchanged
from the beginning till the end of meiosis II (n = 23).

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Prophase II

Spindle fibres reform and attach to centromeres in prophase II.

Metaphase II

The chromosomes align on the metaphase plate during metaphase II in preparation for
centromeres to divide in the next phase.

Anaphase II

In anaphase II, chromosomes divide at the centromeres (like in mitosis) and the
resulting chromosomes, each with one chromatid, move toward opposite poles of the
cell.

Telophase II and Cytokinesis

Four haploid nuclei (containing chromosomes with single chromatids) are formed in
telophase II. Division of the cytoplasm during cytokinesis results in four haploid cells.
Note that these four cells are not identical, as random arrangements of bivalents and
crossing over in meiosis I leads to different genetic composition of these cells.

In humans, meiosis produces genetically different haploid daughter cells, each with 23
chromosomes that consist of one chromatid. These haploid cells become unfertilized
eggs in females and sperm in males. The genetic differences ensure siblings of the
same parents are never entirely genetically identical.

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 Chapter Three:

Body Tissues:

Learning Outcomes

At the end of the instruction the learner should be able to:

 List the four basic tissues of the body

 Describe the characteristics of epithelial tissue
 Describe the various connective tissues
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  Describe characteristics of Muscle tissue
 Describe the Nervous tissue
 State the functions of each of the four tissues

a) Epithelial Tissue:

Epithelial tissue covers the whole surface of the body. It is made up of cells closely
packed and ranged in one or more layers. This tissue is specialized to form the
covering or lining of all internal and external body surfaces. Epithelial cells lining the
inner surface of a structure are referred to as endothelial cells. Epithelial cells are
packed tightly together, with almost no intercellular spaces and only a small amount of
intercellular substance. Epithelial tissue, regardless of the type, is usually separated
from the underlying tissue by a thin sheet of connective tissue; basement membrane.
The basement membrane provides structural support for the epithelium and also binds
it to neighbouring structures.

Types of Epithelial Tissue

Epithelial tissue can be divided into two groups depending on the number of layers of
which it is composed. Epithelial tissue that is only one cell thick is known as simple
epithelium. If it is two or more cells thick such as the skin, it is known as stratified
epithelium.

Simple epithelium

Simple epithelium can be subdivided according to the shape and function of its cells.

 Squamous (pavement) epithelium.

Squamous cells have the appearance of thin, flat plates. The shape of the
nucleus usually corresponds to the cell form and help to identify the type of
epithelium. Squamous cells, for example, tend to have horizontal flattened,
elliptical nuclei because of the thin flattened form of the cell. They form the
lining of cavities such as the mouth, blood vessels, heart and lungs and make
up the outer layers of the skin.

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 Simple squamous epithelium

 Simple Cuboidal Epithelium.

As their name implies, Cuboidal cells are roughly square or Cuboidal in shape.
Each cell has a spherical nucleus in the centre. Cuboidal epithelium is found in
glands and in the lining of the kidney tubules as well as in the ducts of the
glands. They also constitute the germinal epithelium which produces the egg
cells in the female ovary and the sperm cells in the male testes.

Simple Cuboidal epithelium

 Simple Columnar Epithelium

Columnar epithelial cells occur in one or more layers. The cells are elongated
and column-shaped. The nuclei are elongated and are usually located near the
base of the cells. Columnar epithelium forms the lining of the stomach and
intestines. Some columnar cells are specialized for sensory reception such as in
the nose, ears and the taste buds of the tongue. Goblet cells (unicellular glands)

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 are found between the columnar epithelial cells of the duodenum. They secrete
mucus or slime, a lubricating substance which keeps the surface smooth.

Simple columnar epithelium

 Ciliated Columnar Epithelium

These are simple columnar epithelial cells, but in addition, they posses fine
hair-like outgrowths, cilia on their free surfaces. These cilia are capable of
rapid, rhythmic, wavelike beatings in a certain direction. This movement of the
cilia in a certain direction causes the mucus, which is secreted by the goblet
cells, to move (flow or stream) in that direction. Ciliated epithelium is usually
found in the air passages like the nose. It is also found in the uterus and
Fallopian tubes of females. The movements of the cilia propel the ovum to the
uterus.

Ciliated columnar epithelium

 Glandular Epithelium

Columnar epithelium with goblet cells is called glandular epithelium. Some

parts of the glandular epithelium consist of such a large number of goblet cells
that there are only a few normal epithelial cells left. Columnar and Cuboidal
epithelial cells often become specialized as gland cells which are capable of
synthesizing and secreting certain substances such as enzymes, hormones,

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 milk, mucus, sweat, wax and saliva. Unicellular glands consist of single,
isolated glandular cells such as the goblet cells. Sometimes a portion of the
epithelial tissue becomes invaginated and a Multicellular gland is formed.
Multicellular glands are composed of clusters of cells. Most glands are
Multicellular including the salivary glands.

Glandular epithelium

 Stratified Epithelium.

Where body linings have to withstand wear and tear, the epithelia are
composed of several layers of cells and are then called compound or stratified
epithelium. The top cells are flat and scaly and it may or may not be keratinized
(containing a tough, resistant protein called keratin). The mammalian skin is an
example of dry, keratinized, stratified epithelium. The lining of the mouth
cavity is an example of non-keratinized, stratified epithelium.

Stratified epithelium

Functions of Epithelial Tissue

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 Protection: Epithelial cells from the skin protect underlying tissue from
mechanical injury, harmful chemicals, invading bacteria and from excessive loss of
water.

Sensation: Sensory stimuli penetrate specialized epithelial cells. Specialized

epithelial tissue containing sensory nerve endings is found in the skin, eyes,
ears, nose and tongue.

Secretion: In glands, epithelial tissue is specialized to secrete specific chemical

substances such as enzymes, hormones and lubricating fluids.

Absorption: Certain epithelial cells lining the small intestine absorb nutrients from
the digestion of food.

Excretion: Epithelial tissues in the kidney excrete waste products from the body
and reabsorb needed materials from the urine. Sweat is also excreted from the
body by epithelial cells in the sweat glands.

Diffusion: Simple epithelium promotes the diffusion of gases, liquids and

nutrients. Because they form such a thin lining, they are ideal for the diffusion of
gases (such as the walls of capillaries and lungs).

Cleaning: Ciliated epithelium assists in removing dust particles and foreign bodies
which have entered the air passages.

Friction: The smooth, tightly-interlocking, epithelial cells that line the entire
circulatory system reduce friction between the blood and the walls of the blood
vessels.

b) Connective Tissue (supporting tissue)

Characteristics
Connective tissue is divided into subtypes according to the matrix that bind the cells.
Their origin is the mesoderm; they provide structural and metabolic support for other
tissues and organs of the body. Certain types of connective tissue store nutritional
substances while others manufacture protective and regulatory materials.
Supporting tissue occur in many different forms with diverse physical properties. In
most organs, loose supporting tissues act as a biological packing material between

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cells and other tissues with more specific functions .Dense forms of supporting tissue
provide tough physical support in the dermis of the skin, comprise the robust capsules
of organs such as the liver and spleen , and are the source of great strength in ligaments
and tendons.
Cartilage and bone, the major skeletal components are highly specialized forms of
supporting tissue. With the exception of cartilage connective tissue is highly vascular
and well nourished.
The supporting tissue is able to regenerate, and thus functions in the repair of body
organs. All supporting tissues have two major constituents the cells and extracellular
matrix (non-living material), the extracellular matrix is more dominant than the cells.

The cells of the supporting tissue:

 The cells of the supporting tissue may be dividing into several types according
to their functions:
 Cells responsible for synthesis and maintenance of the extracellular material
are derived from precursor cells in primitive supporting tissue
(Mesenchyme).The most common support cell is termed the fibroblast.
 Cells responsible for the storage and metabolism of fat are known as adipocytes
and may collectively form adipose tissue.
 Cells with defence and immune functions are commonly encountered in the
support tissues. These groups of cells include plasma, mast, histiocytes and
tissue macrophages as well as all types of white blood cells. All these cells are
derived from Mesenchyme.

Embryonic Connective Tissue

During the 6-week embryonic period of development all the body organs form. At the
beginning of the embryonic period, all connective tissue look the same and is referred
to as Mesenchyme. This unspecialized connective tissue that consists of irregularly
shaped cells. They later migrate to specific sites and differentiate to form the various
kinds of connective tissue.
Connective Tissue Proper
Connective tissue proper has a loose, flexible matrix called ground substance and is
composed mainly of star-shaped cells called fibroblasts. These cells produce Collagen,
Elastic and Reticular fibres.

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Collagen fibres are composed of protein called collagen; they are flexible and have
tremendous strength.
Elastic fibres are composed of a protein called elastin; it provides some tissues with
elasticity and extensibility.
Reticular fibres are composed of a protein called Reticulin, which is similar in
composition to collagen they form a framework.
There are six basic kinds of connective tissue proper: Loose (Areolar), dense regular,
dense irregular, elastic, reticular and adipose tissues.
Types of Connective Proper
Areolar tissue:
It is characterized by randomly arranged elastic fibre, collagenous fibres and
fibroblasts scattered throughout the matrix.
It is distributed throughout the body, functioning as:
a) Binding and packaging material.
b) Binding the skin to the underlying muscles
c) Providing protection and support to blood vessels and nerves.
It is highly vascular and thus provides nutrients to skin.
Dense regular connective tissue:
\It is characterized by large amounts of closely packed collagenous fibres that run
parallel to the direction of force placed on this tissue during movement.
Found where strong flexible force is required like tendons which attach muscles to
bones and ligaments which connect bones to bones across joints.
Dense irregular connective tissue:
It is characterized by large amounts of closely packed collagenous fibres that are
interwoven to provide strength in any direction.
Found in the dermis of the skin and sub-mucosa of the digestive tract. It also forms the
fibrous covering of organs and joints.
Elastic connective tissue:
The tissue is composed of irregularly arranged elastic fibres and is found in the
trachea, walls of arteries and bronchial tubes.
Reticular connective tissue:
This is characterized by a network of reticular fibres woven through a jellylike matrix.
Some of the cells within the reticular tissue are Phagocytic.
The Liver, Spleen, Lymph nodes and bone marrow contain reticular connective tissue.
Adipose tissue:
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Adipose tissue contains large numbers of adipose cells or adipocytes, these cells store
fat within their cytoplasm causing them to swell and forcing the nuclei to one side.
They are found throughout the body but concentrated, beneath the skin, around the
kidneys, on the surface of the heart and breasts.
Support various organs, keeps the body warm act as food reserve.
Cartilage
It consists of cartilage cells or chondrocytes that occupy tiny spaces called lacunae
within an elastic semisolid matrix. It provides support and protection.
There are three types of cartilage distinguished from one another by the type and
amount of fibres embedded within the matrix. They are Hyaline cartilage,
fibrocartilage and elastic cartilage.
Hyaline cartilage:
Most abundant within the body and is located in the respiratory tract, rib gage and
developing bone.
Fibrocartilage:
It is adapted to withstand tension and compression. It found at the symphysis pubis,
between the vertebra and intervertebral discs and where the two pelvic bones
articulate. Also forms the cartilaginous wedges called menisci within the knee joint.
Elastic cartilage:
It is similar to hyaline cartilage except for abundant elastic fibres which makes it
flexible and strong.
This cartilage is found in the outer ear, portions of the larynx and in the auditory canal.
Bone (Osseous) Tissue
 The bone tissue is the most rigid of all the connective tissue.
 Bone has a rich blood supply and is metabolically very active.
 Has calcium phosphate located in its matrix which is responsible for it
hardness.
 Numerous collagenous fibres give bone some flexibility.
Bone is classified as compact or spongy. The compact bone constitutes the hard outer
portion of the bone; the inner portion is the spongy tissue.
The outer portion is covered by a connective tissue called the periosteum that serves as
a site for attachment of ligaments and tendons, provides protection and gives durable
strength to the bone.
Spongy bone tissue makes the bone lighter and provides a space for the bone marrow
where blood cells are produced.
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The bone cells called Osteocytes are arranged in rings around a central canal
(Harversian) which contains blood vessels and a nerve.
Each osteocyte occupies a space called a lacuna. Radiating from each lacuna are tiny
canals called canaliculi. Nutrients diffuse through the canaliculi to reach each cell.
The matrix layers are called Lamellae.
A central canal with its surrounding Osteocytes, lacunae, canaliculi and concentric
lamellae constitutes an osteon
Blood (vascular) Tissue
Blood as a tissue is a highly specialized connective tissue that plays a vital role in
maintaining internal body homeostasis. It is made formed elements (red blood, white
blood cells and platelets) that are suspended in a liquid matrix the plasma.
Red blood cells are responsible for transportation of oxygen while
White blood cells acts as a defence force to protect the body. On the other hand
Platelets Play a role in the clotting of blood.
c) Muscle tissue:
Structure:
Living organisms can move freely or can perform other types of movement with the
aid of muscles. Muscle tissue has the ability to relax and contract and so bring about
movement and mechanical work in various parts of the body. There are other
movements in the body too which are necessary for the survival of the organism such
as the heart beat and the movements of the alimentary canal.
Muscles can be divided into three main groups according to their structure such as
• Smooth muscle tissue.
• Skeletal muscle tissue.
• Cardiac (heart) muscle tissue.

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Types of Muscle Tissue

• Smooth Muscle Tissue.

Smooth muscle tissue is made up of thin-elongated muscle cell and fibres. These fibres
are pointed at their ends and each has a single, large, oval nucleus. Each cell is filled
with a specialized cytoplasm, the sarcoplasm and is surrounded by a thin cell
membrane, the sarcolemma. Each cell has many myofibrils which lie parallel to one
another in the direction of the long axis of the cell. They are not arranged in a definite
striped (striated) pattern, as in skeletal muscles - hence the name smooth muscle.
Smooth muscle fibres interlace to form sheets or layers of muscle tissue rather than
bundles. Smooth muscle is involuntary tissue; it is not controlled by the brain. Smooth
muscle forms the muscle layers in the walls of hollow organs such as the digestive
tract (lower part of the esophagus, stomach and intestines), the walls of the bladder, the
uterus, various ducts of glands and the walls of blood vessels.
Functions of Smooth Muscle Tissue
• Smooth muscle controls slow, involuntary movements such as the
contraction of the smooth muscle tissue in the walls of the stomach and
intestines.
• The muscle of the arteries contracts and relaxes to regulate the blood
pressure and the flow of blood.

 Smooth Muscle

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 • Skeletal Muscle Tissue.

Skeletal muscle is the most abundant tissue in the vertebrate body. These muscles are
attached to and bring about the movement of the various bones of the skeleton, hence
the name skeletal muscles. The whole muscle, such as the biceps, is enclosed in a
sheath of connective tissue, the epimysium. This sheath folds inwards into the
substance of the muscle to surround a large number of smaller bundles, the fasciculi.
These fasciculi consist of still smaller bundles of elongated, cylindrical muscle cells,
the fibres. Each fibre is a syncytium, which is a cell that has many nuclei. The nuclei
are oval in shaped and are found at the periphery of the cell, just beneath the thin,
elastic membrane (sarcolemma). The sarcoplasm also has many alternating light and
dark bands, giving the fibre a striped or striated appearance (hence the name striated
muscle). With the aid of an electron microscope it can be seen that each muscle fibre is
made up of many smaller units, the myofibrils. Each myofibril consists of small protein
filaments, known as actin and myosin filaments. The myosin filaments are slightly
thicker and make up the dark band (or A-band). The actin filaments make up the light
bands (I-bands) which are situated on either side of the dark band. The actin filaments
are attached to the Z-line. This arrangement of actin and myosin filaments is known as
a sacromere.
During the contraction of skeletal muscle tissue, the actin filaments slide inwards
between the myosin filaments. Mitochondria provide the energy for this to take place.
This action causes a shortening of the sarcomeres (Z-lines move closer together),
which in turn causes the whole muscle fibre to contract. This can bring about a
shortening of the entire muscle such as the biceps, depending on the number of

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muscles fibres that were stimulated. The contraction of skeletal muscle tissue is very
quick and forceful.
Functions of Skeletal Muscle Tissue
• Skeletal muscles function in pairs to bring about the co-coordinated
movements of the limbs, trunk, jaws, eyeballs, etc.
• Skeletal muscles are directly involved in the breathing process.

• Cardiac (Heart) Muscle Tissue.

Cardiac muscle is unique in that it shows some features of skeletal muscle and some
features of smooth muscle. As the name implies, cardiac muscle is the muscle that
makes up the wall of the heart. Cardiac muscle is similar to skeletal muscle in that it is
striated and multinucleate, and similar to smooth muscle in that the nuclei are centrally
located and many cells are required to span the length of the muscle. It differs from
both skeletal muscle and smooth muscle in that its cells branch and are joined to one
another via intercalated discs. Intercalated discs allow communication between the
cells such that there is a sequential contraction of the cells from the bottom of the
ventricle to the top, facilitating maximal ejection of blood from the ventricle during
contraction. This occurs without nervous intervention to each cell or group of cells.
Cardiac muscle also differs from the other two muscle types in that contraction can
occur even without an initial nervous input
The cells that produce the stimulation for contraction without nervous input are called
pacemaker cells
Functions of Cardiac (Heart) Muscle Tissue
• Cardiac muscle tissue plays the most important role in the contraction
of the atria and ventricles of the heart.

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 • It causes the rhythmical beating of the heart, circulating the blood and
its contents throughout the body as a consequence.
d) Nervous Tissue:
Neurons
Neurons are the basic unit of the nervous system. All cells of the nervous system are
comprised of neurons. The nervous system can be divided into two parts: the central
nervous system and the peripheral nervous system. The central nervous system consists
of the brain and spinal cord, while the peripheral nervous system consists of sensory
and motor nervous cells that run throughout the rest of the body. Neurons are
responsible for sending, receiving, and interpreting information from all parts of the
body.
Parts of a Neuron
A neuron consists of two major parts:
Cell body:-Neurons contain the same cellular components as other body cells. The
central cell body is the largest part of a neuron and contains the neuron's nucleus,
associated cytoplasm, and other cell structures. The cell body produces proteins needed
for the construction of other parts of the neuron.
Nerve Processes:
Nerve processes are "finger-like" projections from the cell body that are able to
conduct and transmit signals. There are two types of nerve processes:

Axons - typically carry signals away from the cell body. They are long nerve processes
that may branch out to convey signals to various areas. Some axons are wrapped in an
insulating coat of glial cells called oligodendrocytes and Schwann cells. These cells
form the myelin sheath which indirectly assists in the conduction of impulses as
myelinated nerves can conduct impulses quicker than unmyelinated ones. Axons end at
junctions known as synapses.
Dendrites - typically carry signals toward the cell body. Dendrites are usually more
numerous, shorter and more branched than axons. They have many synapses in order
to receive signal messages from nearby neurons.
Neurons: Nerve Impulse
Axons and dendrites are bundled together into what are called nerves. These nerves
send signals between the brain, spinal cord, and other body organs via nerve impulses.
Nerve impulses are received at the neuronal dendrites and are carried along the axon to
the terminal branches. These branches end at a junction called a synapse. It is at the
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synapse where chemical or electrical impulses must cross the gap and be carried to the
dendrites of adjacent cells. At electrical synapses, ions and other molecules pass
through gap junctions allowing for the passive transmission of electrical signals from
one cell to the other. At chemical synapses, chemical signals called neurotransmitters
are released which cross the gap junction to stimulate the next neuron.
Neuron Classification
Neurons are classified as either motor, sensory, or interneurons. Motor neurons carry
information from the central nervous system to organs, glands, and muscles. Sensory
neurons send information to the central nervous system from internal organs or from
external stimuli. Interneurons relay signals between motor and sensory neurons.

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 Chapter Four:

General pathology

Cell injury and cellular adaptations

Learning outcomes

• At the end of the instruction the learner should be able to:

• Describe etiology of cell injury
• Outline pathogenesis of cell injury
• Classify morphologic forms of cell injury
• Describe the various forms of cell injury
• Explain cellular aging

Introduction

Human body has numerous cells, each of which is assigned predetermined specific
function. The cells form different tissues and cells. In health these cells are in accord
with each other. However, most forms of disease begin with cell injury and
consequently loss of cellular function.

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Cell injury is defined as a variety of stresses a cell encounters as a result of changes in
the internal and external environment.

All cells of the body have in built mechanism to deal with changes in the environment.
The cellular response to changes varies and depends upon the following:

• The type of cell and tissue involved

• The extent and type of cell injury.

Cellular responses to injury may be as follows:

• When there is increased functional demand, the cell may adapt to the changes
which are expressed morphologically and then revert back to normal after the
stress is removed.
• When the stress is mild to moderate, the injured cell may recover (reversible
cell injury) and if the injury is persistent the cell may die (irreversible cell
injury).

The cellular response to changes varies and depends upon the following: The type of
cell and tissue involved and the extent and type of cell injury.

The residual effects of reversible cell injury may persist in the cell as evidence of cell
injury at subcellular level (subcellular changes), or metabolites may accumulate within
the cell.

In order to learn the fundamentals of disease process it is essential to have an

understanding of the causes and mechanisms of cell injury and cellular adaptations. It
is also important to understand the normal human cell, tissues and organs.

Aetiology of cell injury

The causes of cell injury can either be reversible or irreversible and may be caused by
acquired and genetic causes.

Acquired causes are grouped into.

a) Hypoxia and ischemia

b) Physical agents
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c) Chemical and drug agents

d) Microbial agents

e) Immunologic agents

f) Nutritional derangements

g) Psychological factors

In a given situation, more than one of the factors may be involved.

Hypoxia and ischemia

Cells of different tissues essentially require oxygen to generate energy and perform
metabolic functions. Deficiency of oxygen (hypoxia) results in failure to carry out cell
activities.

Hypoxia causes reduced supply of blood to cells (ischemia) and deprivation of oxygen
like in anemia, carbon monoxide poisoning and cardio respiratory- insufficiency.

Physical agents

Physical agents that cause disease include;

 Mechanical trauma like road accidents

 Thermal trauma like heat and cold
 Electric shocks
 Radiations such as ultraviolet and ionizing

Chemical and drug agents

There are many chemicals and drugs that cause cell injury, they include:

 Poisons like cyanide, arsenic and mercury

 Strong acids and alkalis
 Environmental pollutants
 Insecticides and pesticides
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  Oxygen at high concentration
 Hypertonic glucose and salts
 Therapeutic administration of drugs
 Social agents like alcohol and narcotic drugs

Microbial agents

Microbial agents cause infections thus affecting the cells; they include

 Bacteria
 Viruses
 Fungi
 Protozoa and other parasites
 Rickettsia

Immunological agents

Immunity protects the host against various injurious agents but it may also turn lethal
and cause cell injury such as

 Hypersensitivity reactions
 Anaphylactic reactions
 Autoimmune disease

Nutritional derangements

Deficiency or excess of nutrients may result in nutritional imbalances. Nutritional

deficiency diseases may be due to overall deficiency of nutrients as in starvation, lack
of proteins and minerals.

A nutritional excess is a problem resulting in obesity, heart disease and hypertension.

Psychological factors

There is no specific biochemical or morphological changes that are brought about

by psychological factors.

Common acquired mental disease such as mental stress, anxiety, overwork and
frustration may result in depression and schizophrenia.
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 Problems of alcoholism and smoking can result in various organic diseases such
as liver damage and cancer.

Pathogenesis of cell injury

The underlying alterations in biochemical systems of cells for reversible and

irreversible cell injury by varied agents are complex and varied.

However, the following principles apply in pathogenesis of most forms of cell injury.

Type, duration and severity of the injurious agent

The extent of cellular injury depends upon the following:

 The type of stimulus/ injurious agent

 The duration the cell is exposed to the injurious agent
 The severity of the agent to the cell

• Such as small dose of chemical toxin or short duration of ischemia may cause
reversible cell injury while large dose of the same chemical agent or persistent
ischemia cause cell death.

Type, status and adaptability of target cell

• The type of cell as regards its susceptibility to injury, its nutritional status and
adaptation of the cell to hostile environment determine the extent of cell injury.
• Skeletal muscle can withstand hypoxic injury for a long time while cardiac
muscle suffers irreversible cell injury after 30-60 minutes of persistent
ischemia.

Underlying intracellular phenomena

Irrespective of other factors, two essential biochemical phenomena underline all forms
of cell injury to distinguish between reversible and irreversible cell injury.

Inability to reverse mitochondrial dysfunction or reoxygenation

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Disturbance in membrane function in general, and in plasma membrane in particular.

Morphologic consequences

All forms of biochemical changes underlying cell injury are expressed in terms of
morphologic changes.

The ultra-structural changes become apparent earlier than the light microscopic
alterations

The interruption of blood supply (ischemia) and impaired oxygen supply to the tissues
(hypoxia) are most common forms of cell injury in human beings.

Pathogenesis of ischemia and hypoxic injury

They are common causes of cell injury that cause both reversible and irreversible cell
injury.

Reversible cell injury

This occurs as a result of short duration of ischemia and hypoxia. The sequential
changes in reversible cell injury are:

1. Decreased generation of cellular ATP, that is required for a variety of cellular

functions such as membrane transport, lipid synthesis and phospholipid synthesis. ATP
is generated from glucose/glycogen in the absence of oxygen or by oxidative
phosphorylation (requires oxygen). Ischemia and hypoxia both limit the supply of
oxygen to the cells thus causing decreased ATP generation.

2. Reduced intracellular pH. Due to low oxygen supply to the cell, aerobic respiration
by mitochondria fails first. The cell is forced to switch to anaerobic glycolytic pathway
for energy requirement. This results in depletion of glycogen and accumulation of
lactic acid lowering intracellular pH. Intracellular acidosis results in clumping of
nuclear chromatin.

3. Damage to plasma membrane sodium pump. Normally the energy ATP dependent
sodium pump operating at the plasma membrane allows active transport of sodium out
of the cell and diffusion of potassium into the cell. Lowered ATP in the cell interfere

41 | P a g e
with membrane regulated process. This results in intracellular accumulation of sodium
and diffusion of potassium. The accumulation of sodium in the cell leads to increase in
intracellular water to maintain iso-osmotic conditions causing hydropic swelling.

4. Reduced protein synthesis. As a result of continued hypoxia, ribosomes detach from

granular endoplasmic reticulum and polysomes are degraded to monosomes, thus
causing reduced protein synthesis.

5. Functional consequences. Reversible injury to cell may cause functional

disturbance.

6. Ultra-structural changes. Reversible injury to the cell causes the following:

 Plasma membrane: Loss microvilli and local projections of the cytoplasm.

 Mitochondria: Mitochondrial swelling.
 Nucleolus: There is segregation of granular fibrillary components and reduced
synthesis of RNA.
 Endoplasmic reticulum: Detachment of membrane bound polyribosomes from
the surface.

Irreversible cell injury

Persistence of ischemia or hypoxia results in irreversible changes in structure and

function of the cell.

Two phenomena distinguish between reversible and irreversible cell injury.

 Inability of the cell to reverse mitochondrial dysfunction or reperfusion or

reoxygenation.
 Disturbance in cell membrane function in general and in particular plasma
membrane

In addition, there is continued depletion of proteins, leakage of lysosomal enzymes

into the cytoplasm, reduced intercellular pH and further reduction in ATP.

Mitochondrial dysfunction:

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As a result of continued hypoxia, a large cytosolic influx of calcium ions occur
especially after reperfusion of irreversibly injured cell, which is taken by mitochondria
causing dysfunction.

Morphologically, mitochondrial changes seen are vacuoles in the mitochondria and

deposits of amorphous deposits of calcium salts.

Membrane damage:

-Defect in membrane damage function is the most important event in irreversible cell
injury in ischemia. The underlying membrane damages are;

-Accelerated degradation of membrane phospholipids

-Cytoskeletal damage

-Toxic oxygen radicals

-Breakdown products of lipids

-Reperfusion damage

Hydrolytic enzymes:

Damage to lysosomal membranes followed by liberation of lytic enzymes (RNAase,

Protease, glycosidase, Phosphatase and Cathepsins)

These enzymes on activation cause enzymatic digestion of cellular components and

include the nuclear changes (Pyknosis, Karyolysis and Karyorrhesis) and hence cell
death(autolysis).

The dead cell is eventually replaced by masses of phospholipids called myelin figures
which are either phagocytized by macrophages or there may be formation of calcium
soaps (deposits).

Serum estimation of liberated intracellular enzymes:

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Liberated enzymes already mentioned leak across the abnormally permeable cell
membrane into the serum, the estimation of which may be used as clinical parameters
of cell death.

Depending on the duration of ischemia/hypoxia, restoration of blood flow may result

in the following consequences:

 When the period of ischemia is short, reperfusion with re-supply of oxygen

restores the structural and functional state of the injured cell (reversible injury)
 When ischemia is of longer duration, reperfusion deteriorates the already
injured cell (ischemia-reperfusion injury)
 Longer period of ischemia may also produce irreversible cell injury during
ischemia itself without any role of reperfusion. This is due to marked
intracellular excess of sodium and calcium ions due to cell membrane damage.

Pathogenesis of chemical injury:

Chemicals induce cell injury by one of the following two mechanisms:

1. Direct cytotoxic effects. Some chemicals combine components of the cell and
produce direct cytotoxity without requiring metabolic activation.

The cytotoxic damage is usually greatest to cells which are involved in the metabolism
of such chemicals such as in mercuric chloride poisoning, the greatest damage is to
cells of the alimentary tract and kidney. Examples of direct cytotoxic chemicals
include, chemotherapeutic agents used in treatment of cancer, toxic heavy metals such
as mercury, lead and iron. Cyanide kills the cells by poisoning mitochondrial
cytochrome oxidase thus blocking oxidative phosphorylation.

2. Conversion to reactive toxic metabolites. This involves metabolic activation to

yield ultimate toxin that interacts with the target cell. The target cell in this group
of chemicals may not be the same cell that metabolized the toxin. Example of cell
injury by conversion of reactive metabolites is toxic liver necrosis caused by
carbon tetrachloride and bromo-benzene.

Pathogenesis of physical injury:

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Injuries caused by mechanical force are of medical legal significance. They may lead
to state of shock.

Radiation injury to human by accidental or therapeutic exposure is of importance in

treatment of persons with malignant tumors as well may be carcinogenic.

Ionizing radiations kill cells as a result of direct formation of hydroxyl radicals from
radiolysis of water. These radicals damage the cell membrane and may interact with
DNA of target cell inhibiting DNA replication and eventual cell death.

Morphology of cell injury:

Depending on severity of cell injury, degree of damage and residual effects of cells and
tissues are variable. Microscopic morphologic changes in various forms of cell injury
are classified in various forms;

Morphology of reversible cell injury

Degeneration of cells has been used to denote reversible cell injury. Currently
retrogressive changes or reversible cell injury are used for non-lethal cell injury.
Following morphologic forms of reversible cell injury such changes include:

Cellular swelling (Hydropic change, vacuolar degradation):

This is the commonest and earliest form of cell injury from all cases. The common
causes of cellular swelling include; bacterial toxins, chemical, poisons, burns, high
fever, intravenous administration hypertonic glucose. The swelling is as a result of
influx of sodium and extracellular water into the cell and escape of potassium. Small
vacuoles are seen in the cell hence the term vacuolar degradation.

Hyaline change:

The word hyaline means glassy in appearance. This is therefore a term used to describe
homogenous, eosinophilic appearance of material in Haematoxylin in eosin stained
sections. It is associated with pathologic conditions and may be intra or extracellular.
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Examples are; nuclear or cytoplasmic inclusions in some viral infections and Russell
bodies due to excessive immunoglobulins in the rough endoplasmic reticulum of the
plasma cells.

Fatty change:

This is the intracellular accumulation of neutral fat within parenchymal cells. The
deposit is in the Cystosol and is common in the liver but may occur in other non-fatty
tissues like the heart and skeletal muscle. The commonest cause of fatty liver include;
starvation, malnutrition, obesity and excess alcohol consumption.

Classification of morphologic forms of cell injury

Mechanism of cell injury Nomenclature

1. Reversible cell injury Retrogressive changes

(degenerations)

2. Irreversible cell injury Cell death- Necrosis

3. Programmed cell death Apoptosis

4. Residual effects of cell injury Subcellular alterations

5. Deranged cell metabolism Intracellular

accumulation of lipid,

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 protein and carbohydrates

6. After effects of necrosis Gangrene, pathologic

calcification

Intracellular accumulations:

Intracellular accumulation in abnormal amounts can occur within the cytoplasm or

nucleus of the cell. This phenomenon was previously referred to as infiltration.

Abnormal accumulation can be divided into:

-Accumulation of constituents of normal cell metabolism.

-Accumulation of abnormal substances.

-Accumulation of pigments.

Morphology of irreversible cell injury:

Cell death is a state of irreversible injury. It may occur in the living body as a local or
focal change as a result of autolysis, necrosis and apoptosis and the change that follow
it like gangrene and pathologic calcification or result in end of life (somatic death).

Autolysis:

This is the disintegration of the cell by its own hydrolytic enzymes liberated from the
lysosomes. Autolysis can occur in the living body when it is surrounded by
inflammatory reaction or may occur as a postmortem change. Autolysis is rapid in
some tissues rich in hydrolytic enzymes such as the pancreas and gastric mucosa but
slow in fibrous tissue. Morphologically autolysis is identified by homogeneous and
eosinophilic cytoplasm with loss of cellular details and remains of cell as debris.

Necrosis:

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It is defined as focal death along with degradation of tissue by hydrolytic enzymes
liberated by cells. Necrosis can be caused by agents such as hypoxia, chemical and
physical agents, microbial agents and immunological injury. Irreversible cell injury in
necrosis is brought about by cell digestion by lytic enzymes and denaturation of
proteins.

The cytoplasm appears homogenous and intensely eosinophilic. Occasionally it may

show vacuolation or dystrophic calcification.

The nuclear changes include condensation of nuclear chromatin (Pyknosis), dissolution

of nuclear chromatin (Karyolysis) or fragmentation into many granular clumps
(Karyorrhesis).

Necrosis can be divided into:

Coagulative necrosis: Most common type is caused by ischemia and less often from
bacteria and chemical agents. Most affected organs are heart, spleen and kidney.

Liquefication necrosis: Occurs mostly due to ischemia, bacteria or fungal infections

Gaseous necrosis: Found in the centre of foci of tuberculosis infection.

Fat necrosis: It is a special form of cell death at two anatomically different locations
but morphologically similar lesions. It is Common in acute pancreatic necrosis and
traumatic fat necrosis of breasts.

Fibrinoid necrosis: It is characterized by deposition of fibrin-like material. It is

encountered in various immunological injury e.g. autoimmune diseases.

Apoptosis:

It is a form of coordinated and internally programmed cell death which is of

significance in a variety of physiologic and pathologic conditions.

Some of the changes that occur in cells in apoptosis include; shrinkage of cells,
chromatin condensation around the edge of the nucleus and Phagocytosis of apoptotic
bodies by macrophages.

Cellular adaptations:
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For the sake of survival on exposure to stress, the cells make adjustments with the
changes in their environment to the physiological needs and to non-lethal pathological
injury. These physiological and pathological adaptation occur by the following
processes:

 Decreasing or increasing in size i.e. atrophy and hypertrophy or increasing the

number i.e. hyperplasia.
 By changing the pathway of phenotypic differentiation of cells i.e. Metaplasia
and dysplasia.

Various mechanisms which may be involved in adaptive cellular responses

include:

• Altered surface receptor binding

• Alterations of protein synthesis
• Synthesis of proteins by target cell such as heat-shock proteins.

Atrophy:

This is the reduction of the number and the size of cells of an organ or its parts which
was once normal. Atrophy is caused as physiologic or pathologic atrophy.

Physiologic atrophy is a normal aging process in some tissues which could be due to
loss of endocrine stimulation or arteriosclerosis.

Pathologic atrophy is caused by various factors such as; starvation, ischemia, loss of
endocrine regulatory mechanism and prolonged diminished functional activity of the
cell or organ.

Irrespective of the underlying causes of atrophy the pathologic changes are similar.
The organ is small, often shrunken. The cells become smaller in size but are not dead.
Shrinkage in cell size is due to reduction in cell organelles e.g. mitochondria,
myofilaments and endoplasmic reticulum, vacuoles may be present containing cell
debris such as Lipofuchsin pigment.

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Hypertrophy:

This is an increase in the size of cells resulting in enlargement of the organ or tissue
without any change in the number of cells. Hypertrophy may be physiological or
pathological and in both cases may be caused by increased functional demand or by
hormonal stimulation. An example of physiologic hypertrophy is enlargement of
uterus during pregnancy.

An example of pathological hypertrophy includes; hypertrophy of cardiac muscle

which may occur in a number of cardiovascular diseases and hypertrophy of smooth
muscle like in muscular arteries in hypertension.

The affected organ is enlarged and heavy. At ultra- structure level there is increased
synthesis of DNA and RNA, increased protein synthesis and increased number of
organelles.

Hyperplasia:

This is the increase in the number of cells resulting in enlargement of the organ/tissue.
Quite often, both hyperplasia and hypertrophy occur together. Hyperplasia occur due
to increased recruitment of cells from Go (resting) phase of the cell cycle to undergo
mitosis.

As other disorders hyperplasia has also been divided into physiological and
pathological.

Metaplasia:

Meta= means transformation, plasia=growth. It is defined as a reversible change of

one type of epithelial or mesenchymal adult cells to another type of adult epithelial or
mesenchymal cells. This is in response to an abnormal stimulus. If the stimulus
persists for a long time the epithelium may become cancerous.

Epithelial Metaplasia can result in replacement of normal epithelial layer with a less
specialized one.

Dysplasia:

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It means disordered cellular development, often accompanied with Metaplasia and
hyperplasia. It is also referred to as atypical hyperplasia. The condition mostly occur
in epithelial cells and is characterized by cellular proliferation and cytologic changes
that include;

-Increased number of layer of epithelial cells.

-Disorderly arrangement of cells from basal layer to the surface layer.

-Loss of basal polarity i.e. nuclei lying away from basement membrane.

-Cellular and nuclear pleomorphism.

-Increased nuclear/cytoplasmic ratio.

-Nuclear hyperchromatism.

-Increased mitotic activity.

Most common examples of dysplastic changes include; uterine, cervix and respiratory
tract.

Dysplastic changes often occur due to chronic irritation or prolonged inflammation.

Removal of inciting stimulus, the changes may disappear.

In a proportion of cases, however, dysplasia progress into insitu (cancer confined to

layers superficial to basement membrane).

Cellular aging:

Old age is a concept of longevity in human beings. The consequences of aging appear
after reproductive age. Aging is distinct from mortality and disease although aged
individuals are more vulnerable to disease.

The life expectancy of an individual depends upon the following factors;

 Intrinsic genetic process; the gene controlling endogenous and exogenous

factors initiate apoptosis. Environmental factors e.g. consumption and
inhalation of harmful substances, diet and role of antioxidants.
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  Lifestyle of the individual such as diseases due to alcoholism, smoking and
drug addiction.
 Age related diseases e.g. atherosclerosis and ischemia, heart disease, diabetes
hypertension and Parkinson's disease.

Cellular basis:

With age, structural and functional changes occur in different organs and systems of
the human body. There is no definitive biologic basis of aging is established, most
acceptable theory is the functional decline of non-dividing cells such as neurons.

The following hypothesis based on investigations explains the cellular basis of aging;

 Genetic controlling invertebrates.

 Clock genes responsible for controlling the rate and time of aging have been
identified in lower invertebrates such as mutation gene in the metazoa results in
prolonging the lifespan of the worm and slowing some of the metabolic
functions.

Diseases of accelerated aging:

Aging is under genetic control in human beings supported by observation of high

concordance in lifespan of twins. A condition associated with signs of accelerated
aging process is termed progeria and is characterized by baldness, cataract, sand
coronary artery disease and grey hair.

Another example is Werner syndrome, a rare autosomal recessive disease

characterized by similar features of premature aging, atherosclerosis and risk of
development of various cancers.

Oxidative stress hypothesis:

Currently, it is believed that aging is partially caused by progressive and reversible

molecular oxidative damage due to persistent oxidative stress on the human cells. In
normal cells, very small amount (3%) of total oxygen consumption by the cell is
converted in reactive oxygen species. The generation of reactive oxygen species is

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directly correlated with metabolic rate of the organism. With aging, there is low
metabolic rate with generation of oxygen radicals which fail to get eliminated causing
their accumulation resulting in cell damage. The underlying mechanism appears to be
oxidative damage to mitochondria.

Chapter Five:

Fixation:
Learning outcomes
At the end of the instruction the learner should be able to:
 Describe fixation process
 State the characteristics of the various fixatives
 Describe classification of fixatives
 Outline the process of fixing gross specimens
 State treatment and storage methods of fixed tissues

Introduction:
The process of using chemical substance (solutions) to preserve tissues is known as
fixation and the chemicals are known as fixatives.

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The aims of fixation is to stop post mortem changes by preserving the structures,
shape, relationship and constituent of tissues and cells in as life like manner as
possible.
The objective of fixation is to maintain clear and consistent morphological features. In
order visualize micro-anatomy of tissues, its stained sections must maintain the
original microscopic relationships among cells, cellular components (like cytoplasm
and nuclei), and extracellular material with little disruption of the organization of the
tissue and must maintain the local chemical composition
Fixation therefore is the foundation of processing of tissues for diagnosis of diseases,
research learning, museum exhibition and monitoring of disease treatment.
Many tissue components are soluble in aqueous acid or other liquid environments, and
a reliable view of microanatomy and microenvironment of these tissues requires that
the soluble components are not lost during fixation and tissue processing.
For example, if soluble components are lost from the cytoplasm of cells, the colour of
the cytoplasm on haematoxylin and eosin staining will be reduced or modified thus
evaluations of structure and function will be reduced or lost.
Fixation of tissues can be accomplished by physical and/or chemical methods. Physical
methods include heating, microwaving and freeze drying. Most histological fixation
methods use chemical liquid fixatives.
The result of all subsequent procedures will depend on the correct selection and use of
fixatives. Good results can be obtained by considering the following:-
i) Structures to be demonstrated must be taken born in mind.
ii) Tissues must be fixed as soon as possible.
iii) Amount of fixatives must be 15-20 times the size of the tissue.

Fixatives
These are chemical solutions that will preserve after death the shape, structure,
relationship and constituent of tissues and cells in as life like manner as possible after
removal from the body.
There are several fixatives that are available but their use depends on.
 The type of tissue.
 Urgency of investigation
 Tissue structures required for demonstration and its side effects both to
tissue and user.
 Type of straining procedure.
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Qualities of a good fixative:
1. It must kill the tissue cells quickly (to avoid further metabolism).
2. Must inhibit autolysis by inactivating enzymes.
3. Must stop Putrefaction (anti-bacterial).
4. Must render soluble substances of the cell insoluble.
5. Must penetrate the cell quickly and evenly.
6. Must harden the tissue and enable easy manipulation of Sgt tissue.
7. Must alter to varying degrees the refractive indices of different cell structures
so that they are made visible due to differentiation.
8. It must fortify the tissue against the harsh effects the solution used during
processing.
9. It must have a good effect on strain.
10. Easy to prepare and not expensive in terms of cost
Effects of fixatives:
Fixatives have the following effects
a) Precipitation: Fixatives make soluble substances of the cells and tissues
insoluble by precipitating or coagulating the soluble proteins; this will it hard
and therefore allow manipulation of soft tissues
b) Inactivation of enzymes: Enzymes bring about metabolic activities within the
cells and this may interfere with the characteristics of the tissue structures,
fixatives are therefore used to inactivate them. The importance of fixation is to
make sure that there is no change that takes place after removal or death of
tissue hence the need to stop the action of enzymes. When the cells die the
lysosomes rapture thus releasing lytic enzymes that digest the surrounding
tissue, inactivation of enzymes is important to avoid such effects.
c) Anti- bacterial: Like the enzymes bacteria can cause distortion of the tissue
structures resulting of the rotting of tissue. Bacteria that may be present as
commensals, from the environment or may have brought about disease may
continue multiplying bringing certain changes in the tissue. The fixatives have
the ability to kill such bacterial cells.
d) Increase of refractive index: Certain fixatives increase or raise the refractive
indices. This will allow the various tissue structures to be clearly seen during
microscopic examination.
e) Introduction of pigments: Certain fixatives when used can impart unwanted
pigments onto tissues; this may mask the tissue structures to be examined
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 microscopically. The pigment could be oxides as a result of chemical reactions
between the fixatives and chemical constituents of tissue or due to the colour of
the fixative used.
f) Health hazards: Some fixatives can bring about harmful effects to the user.
Formaldehyde for example when handled carelessly may cause sinusitis and
dermatitis, while Osmic acid fumes may result in blindness if in contact with
eye tissue.
g) Swelling: Some fixatives may make the tissue swell that may be mistaken to be
an abnormality during examination of tissue.
h) Shrinkage: A large number of tissues may shrink the tissue to an extent that
wrong diagnosis may be given, the tissue may be very normal but due to its
shrunken state it look abnormal.
i) Mordanting effect: Certain fixatives will give improved preservation and
staining of some tissue structures, such fixatives may be used as secondary or
after initial fixation in another fixative.
No single fixative can have all the qualities outlined above so it is necessary to have a
knowledge of wide range of fixatives in order to be able to select the most suitable
fixatives as the occasion demands fixatives are usually divided into two types:-
a) Simple fixatives.
b) Compound fixatives.
Simple fixatives:
These are fixing solutions that are made up of one chemical fixing agent they include
- Formaldehydes.
- Mercuric chloride
- Potassium Dichromate
- Osmium Tetroxide ( Osmic acid)
- Picric acid
- Chromic acid
- Acetone
- Trichloacetic acid.
- Ethyl alcohol
- Acetic acid.
1. Formaldehyde (HCHO)
- It is a gas produced by oxidation of methyl alcohol. It is soluble in H 2O to
an extent of 40% by weight.
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 - Preserves fats and Mucins but without precipitating them
- It is a powerful reducing agent
- Formaldehyde after prolonged storage develops a white deposit known as
Para-Formaldehyde. This deposit can be prevented by storing at low
temperature though its presence does not impair the fixing qualities.
- It is usually acidic due to presence of formic acid. This acid can be
neutralized by addition of magnesium carbonate or sodium hydroxide.
2. Mercuric Chloride (HgCl2)
- It is mostly used in conjunction with other fixatives and usually in aqueous
saturated solution.
- Precipitate all proteins but doesn’t combine with them.
- Hardens and shrinks the tissues
- Poisonous and also corrosive to metals
- Penetrate the tissue fairly quickly.
- Leaves a black precipitated on tissue know as mercuric chloride pigment
which can be removed by lugol’s iodine method.
3. Potassium Dichromate ( K2Cr2O7).
- Forms an insoluble lower oxide which can’t be removed in tissues.
- Fixes different tissue structures at different PH e.g. cytoplasm and
mitochondria at PH 3.4 -3.8.
- Precipitate proteins and preserves carbohydrate.
- Tissue becomes brittle on long fixation.
- Any tissue fixed in it must be washed thoroughly in running tap water.
4. Chromic acid (CrO3)
- It is a powerful oxidizing agent.
- Precipitate proteins and preserves carbohydrates.
- Tissues must be washed thoroughly after fixation.
5. Osmium Tetroxide (OSO4). (Osmic acid).
- Has a dangerous (poisonous) and irritant vapour.
- Demonstrate lipids by fixing and straining them black.
- Penetrates tissues unevenly and poorly.
- Easily reduced by light, warmth or any organic matter so that it must be
kept in dark and cool place.
- Can cause blindness when deposited in the eyes

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 - The only fixative that fixes fats permanently.
6. Picric Acid C 6H2(O2)3OH
- A chemical that can be used as a fixatives stain and differentiator. It can also be used
to remove formalin pigment and malarial pigment.
- It can explode if left to dry and must be kept damp by storing under water.
- Precipitate proteins by forming picrates which are water soluble. These picrates must
be made insoluble by treating with alcohol.
- Causes much shrinkage but little hardening on tissues.
7. Glacial Acetic acid ( CH3COOH)
- Colourless solution with pungent.
- Solidify at 17ºC if water free.
- Swells collagen fibres and precipitates proteins.
- Dissolves some cytoplasmic granules e.g. mitochondria and golgi apparatus
8. Trichloracetic acid ( CCI3COOH)
- General protein precipitant but has marked swelling on most tissues
- Can be used as decalcifying agent.
9. Ethyl Alcohol ( C2H2OH2)
- Used as a fixative at a concentration of between 70-1--% with a reducing
agent.
- Is a reducing agent
- Preserves glycogen but pre dissolve most lipids.
- Causes little shrinkage and hardening.
10. Acetone:
- Used as cold acetone at 4oC to fix enzymes particularly phosphates and
lipase.
Compound fixatives:
These are chemical substances (solutions) which are made up of two or more
simple fixatives
Simple fixatives are mixed together to obtain their combined effect on tissue.
Compound fixatives are divided into two:-
- Micro -anatomical fixatives
- Cytological fixatives

Micro-anatomical fixatives
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These are fixatives that are used for processing various types of tissues and cells in
relation to one another, so that their general structure can be studied.
There are several micro anatomical fixatives they include:-
a) 10% formalin or 10% formal saline (can be buffered or not).
b) Formal sublimate.
c) Susa Heidenhain’s
d) Zenker’s fluid
e) Helly’s fluid
f) Bouin’s fluid
10% Formalin:
This is a simple fixative it can be prepared as 10% formal saline or 10% neutral
buffered formalin or 10% buffered neutral formal saline.
10% formalin composition:
It is composed of 40% formaldehyde (100ml) and water (900ml).
10% formal saline composition
o It is composed of :-
o 40% formaldehyde -100ml
o Sodium chloride – 8.5 gm
o Water - 900ml
Mode of preparation:
1. First prepare normal saline
2. Then use the formula Required concentration X Required volume
Original concentration
-The two fixatives function on the same principle except 10% formalin does not
preserve red blood cells while 10% formal saline does it.
-They impart a firm consistency to tissue without excessive hardening.
-Does not have any effect on tissues on long fixation.
-They are the only fixatives that can allow restoration of natural colour of tissues
therefore is useful in preservation of museum specimens carried on tissues.
-Fixation time is 24-48 hours.
-Blood containing tissues show some pigment when fixed in the fixative, this pigment
is known as formalin or post-mortem pigment.
-On long standing they form formic acid which can be prevented by addition of
“Marble chips” (Calcium carbonate).

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-It can cause dermatitis and sinusitis on long usage.
10% Buffered formalin.
1. It is composed 40% formaldehyde -100ml
2. Water -900ml
3. Acid sodium phosphate monohydrate ( Na2H2PO4H2O)
4. Anhydrous disodium phosphate (Na2HPO4) – 6.5 grams.
5. Sodium chloride – 8.5grams
When it is without sodium chloride it is known as 10% buffered formalin but when it
has sodium chloride it is known as buffered formal saline.
All its properties are the same as those of formalin or formal saline except there is no
formation of formic acid.
Mercuric chloride formalin (formal sublimate)
Composition
1. Composed of saturated aqueous mercuric chloride - 900ml
2. Formalin -100ml
-Excellent micro anatomical fixative
-It is used as a secondary fixative as it enhances staining reactions with acid dyes and
metachromatic stains
-Good for preservation of nervous system especially the myelin sheath tissue.
-Fixation time is 12-24 hours.
-It shrinks the tissue without distortion.
-It is corrosive and expensive.
-Imparts mercuric chloride pigment on tissues.
Heidenhain’s Susa
Composition
a) Mercuric chloride – 45grams
b) Sodium chloride - 5grams
c) Acetic acid -40grams
d) Formalin – 200 ml
e) Water - 800ml
- Fixation time takes 12-24 hours.
- It is a general purpose fixative for routine work.
- Has poor preservation of Red blood cells
- Particularly for post-mortem materials.
- Gives brilliant staining results.
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 Zenker’s fluid
Composition
1. Potassium dichromate - 2.5grams
2. Mercuric chloride - 5grams
3. Sodium sulphate - 1.0gram
4. Water- 100ml
5. Acetic acid - 5ml (Add just before use.)
- The stock solution keeps well without acetic acid.
- Most useful in fixation of fresh tissues than post- mortem material
- Fixation time 3 -18 hours
- Good for connective tissue fibres
- Tissues should be washed thoroughly in water after fixation to remove colouration by
potassium dichromate.
- Tissue becomes brittle on long fixation.
- It leaves mercuric chloride pigment on tissues.
Helly’s fluid
Composition
1. Potassium dichromate – 2.5grams
2. Mercuric chloride -5grams
3. Sodium sulphate - 1.0gram
4. Water - 100 ml
5. Formaldehyde – 5 ml (added just before use)
- Fixation time 6 -24 hours.
- Used as secondary fixative
- Suitable for bone, marrow, spleen, lymph glands, pancreas and pituitary glands
- Tissues become brittle on long fixation.
Bouin’s fluid
Composition:
1. Saturated aqueous picric acid – 75ml
2. 40% formaldehyde -25 ml
3. Acetic acid (Ethanoic acid) – 5ml.
- Fixation time 6-24 hours
- Makes collagen fibres swell
- Causes complete lysis of Red blood cells
- Permit brilliant staining results.
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- No excessive hardening of tissues
- Poor penetration so it is recommended for small pieces of tissue
Cytological fixatives:
These are fixatives that are for preservation of constituent element of cells.
They are divided into two:-
1. Nuclear fixatives
2. Cytoplasmic fixatives.
1. Nuclear Fixatives:
These are fixatives that preserve the structures within the nucleus, they include:
- Fleming’s fluid
- Carnoy’s fluid
- Clark’s fluid
- Sanfelice fluid
a) Fleming’s Fluid.
- 1% chromic acid – 15 ml
- 2% osmium tetroxide - 4ml
- Acetic acid - 1 ml
-It is prepared just before use.
-Fixation time 2 -3 days.
-Used as a secondary fixative.
-Causes excessive blackening of superficial layers due to presence of osmium
tetroxide.
b) Carnoy’s Fluid.
1. Absolute alcohol – 60ml
2. Chloroform - 30 ml
- Fixation time is 30-90 mins
- Good for urgent biopsies
- Dissolve some cytoplasmic granule.
- Causes excessive shrinkage.
c) Clark’s fluid.
1. Absolute ethanol -75 ml
2. Acetic acid - 25 ml
Fixation time - 15 minutes
Good fixative for chromosomes

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d) Sanfelice
– Solution A- Formalin
- Acetic acid
-Solution B -1% chromic acid
Good for fixation of chromosomes and mitotic phases
2. Cytoplasmic Fixatives:
These are fixatives that preserve structures in the cytoplasm of the cell
They include:-
a) Muller’s fluid
b) Orth’s fluid
c) Schauddin’s fluid.
a) Muller’s fluid
1. Potassium dichromate - 2.5 grams
-2. Distilled water- 100ml
-Fixation time is 24-48hrs
-Has mordanting effect on tissues thus improving on preservation and staining
b) Orth’s fluid
1. Formalin -10ml
2. Muller’s fluid -100ml
-Does not keep well and should be prepared just before use
c) Schauddin’s fluid
1. saturated aqueous mercuric chloride - 2parts
2. Absolute ethanol - 1part
-Best for fixing smears
-Fixation time is 15 minutes
d) Sanfelice
Solution A – Formalin
-Acetic acid
Solution B- 1% chromic acid
Vapour fixation:
A form of fixation where the tissue does not come in contact with the fixative directly,
but it is fixed using vapour from heated liquid fixatives used .They are used for very
delicate tissue structures such as fats and enzymes, formaldehyde is one such fixative.
The tissue is suspended and not touching the fixative then a little heat is applied, the
vapour then comes in contact with the tissue fixing it.
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Treatment of tissues after fixation
After fixation in various fixatives, tissues are treated as follows
1. Tissues fixed in 10% formal saline is transferred to 70% alcohol
2. Bouin’s fluid - 70% alcohol
3. Formal sublimate - 80-90% alcohol
4. Heidenhain’s Susa - 95% alcohol
5. Carnoy’s fluid - 95% alcohol
6. Helly’s fluid - wash in running tap water for 12-24 hrs
7. Zenker’s fluid - wash in running tap water for 12-24 hrs
8. Flemings fluid - wash in running tap water for 12-24 hrs
9. Orth’s fluid - wash in running tap water for 12-24 hrs
10. Sanfelice fluid - wash in running tap water for 12-24 hrs
Storage of tissues after fixation
After fixation tissues can be stored in the following solutions for any length of
time
1) 10% formalin, formal saline (buffered)
2) 70% alcohol
3) 30% glycerol
4) 10-20% diethylene glycol
5) 1% phenoxerol
6) Paraffin wax block
The storage is done for the following reasons
i. Future reference
ii. Research work
iii. Teaching purposes
iv. When results are not required immediately
v. General demonstration like museum ( Exhibitions)
Fixation of gross specimens (whole organs)
Fixation of large specimens including whole organs is done in different ways
depending on the type of specimen. The volume of the fixative in which the tissue will
be finally immersed should be 50-100 times the size of the tissue.
Fixation can be done for a period of 2 weeks.
Organs that can be fixed whole include: Brain, kidney, heart, liver and eye.
The method used to fix them is referred to as perfusion and immersion.
Perfusion
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  Wash the organ in 10% formal saline to remove blood clots
 Inject 10% formal saline using a syringe into a major blood vessel and let it to
run out of the organ until no more blood clots are seen.
 Therefore tie both ends of the blood vessel bearing some fixation in it.
Immersion
Immerse the organs in the fixatives until it is wholly covered. The volume of the
fixative should 50-100 times the size of the tissue.
Fixation of elongated tissues:
Muscle:
Stretch gently onto a white board and allow it to adhere to the board for a few minutes
then immerse the board vertically in the fixative. The muscle can also be placed on the
board and both ends are pinned onto the board then inserted vertically into the fixative.
The muscle can also be suspended in the fluid using a support with some weight
attached at the bottom.

Muscle fixed on a cardboard

Muscle

Cardboard
Pin

Muscle suspended in fixative

Muscle

Fixative Weight

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Spinal cord:
Open up the spinal cord and chop into small pieces or lengths. Pin it out flat on a
wooden support and placed in a tall cylindrical fix.
Nerves:
Place on a white cardboard and keep it straight to get longitudinal and transverse
sections and immerse in the fixative
MUSEUM TECHNIQUES
Introduction:
All teaching hospitals and colleges of Pathology have Museums which serve
Many functions: permanent exhibition of common specimen for undergraduate and
postgraduate teaching purposes, illustrating specimens of rarity, permanent source of
histologic material and for gross and microscopic photography.

Objectives
After reading this lesson, you will be able to:
 Explain the methods used in handling museum specimens
 Describe the techniques of specimen preservation.
Basic museum techniques
Any specimens for museum are handled by following steps:
1. Reception2. Preparation
3. Fixation
4. Restoration
5. Preservation
6. Presentation
Reception of the Specimen
Any specimen received in the museum should be recorded in a Reception book and
given a number followed by year (e.g. 32/2013). This number will stay with the
specimen even after it is catalogued in its respective place. This number is written on
tie-on type label in indelible ink and is firmly attached or stitched to the specimen. The
reception book should contain all necessary information about the specimen (clinical,
gross and microscopic findings).
Preparation of the specimen

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An ideal specimen is received fresh in unfixed state. However, it is mostly obtained
from pathology laboratory after being examined, thus will already be formalin fixed. If
planning to use a specimen for museum, part of it can be kept without disturbing for
museum, e.g. in kidney it can be bisected and one half kept aside for museum.
Fixation of the specimen
The objective of fixation is to 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. Fixation arrests autolysis and bacterial decomposition and stabilizes
the cellular and tissue constituents. The fixatives used in museums all over the world
are based on formalin fixative technique, and are derived from Kaiserling technique
and his modifications. Kaiserling recommended that the initial fixation be a neutral
formalin (KI) solution and then transferred to a final preserving glycerine solution
(KIII) for long term display. Colour preservation is also maintained with these
solutions.
Kaiserling’s Technique
Fixation of specimen:
The specimen needs to be kept in a large enough container which can accommodate
specimen along with 3-4 times volume of fixative. Specimen is stored in the Kaiserling
I Solution for 1 month depending on the size of the specimen. The specimen should
not rest on bottom or an artificial flat surface will be produced on hardening due to
fixation.
Kaiserling I Solution:
Formalin 1L
Potassium acetate- 45 g.
Potassium nitrate- 25 g.
Distilled water Make up to 10 litres
Restoration of specimen
It is required to restore the specimens, as they lose their natural colour on fixation.
The recommended method is the Kaiserling II method. It involves removing the
specimen, washing it in running water and transferring to 95% alcohol for 10 minutes
to 1hour depending on the size of specimen. The specimen is then kept and observed
for colour change for around 1- 1.5 hrs. After this step, specimen is ready for
preservation.
Kaiserling II Solution:
Alcohol 95%
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Store specimen in this solution for- (10 min- 1 hour), depending on size of organ.
Rejuvenator Solution:
Pyridine 100 ml
Sodium hydrosulphite 100 gm
Distilled water 4 litres
Formalin decreases the natural colour of the specimen. However, rejuvenator solution
restores the colour.
Preservation of specimen
The recommended solution for this step is Kaiserling III. This is the final solution in
which the specimen will remain for display. It is based on glycerine solution.
Kaiserling III Solution:
Potassium acetate
Glycerine 4 litres
Distilled water Make up to 10 litres
Thymol crystals added to prevent moulds.
Leave solution to stand for 2 – 3 days before using to ensure proper mixing of
chemicals.
Add 1% pyridine as stabilizer
This solution acts as permanent fixative. This solution easily turns yellowish and needs
to be replaced to restore colour of the specimen. The specimen will initially float to
surface but later sink to bottom.
Presentation of the Specimen
Initially all museum specimens were mounted in cylindrical jars and sealed with sheep
bladder walls. Later they were replaced by rectangular glass jars. They were better than
cylindrical ones as the flat surfaces afforded a clear view of specimens without any
distortion. They are covered by rectangular glass plates.
These jars can be purchased readymade or assembled in museum itself, as per need.
Nowadays, Perspex jars are also available, which are lighter than glass jars.
However, they cannot be used to store specimens fixed in alcohol or methyl salicylate
as they react with plastics.
Mounting the Specimens
To support the specimen within its jar, it is attached to the specimen plate or
rectangular bent glass rods. It can be done by tying the specimen with nylon threads.
Double knots should be made by threads, on the specimen surface.

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Factors affecting the quality of fixation:
Buffers and pH
The pH and buffering of fixatives is very important since various tissue structures
respond to fixation based on hydrogen ion concentration (H+) and hydroxyl groups
(OH-)
Duration of fixation and size of specimen
The depth of penetration of the fixative into the tissue is dependent on the duration of
exposure and the size of tissue.
Temperature of fixation
The diffusion of molecules increases with rising temperature due to their more rapid
movement and vibration like the rate of penetration of a tissue by formaldehyde is
faster at higher temperatures. Microwaves therefore have been used to speed fixation
both by increasing the temperature and molecular movements.
Concentration of the fixative
Effectiveness and solubility primarily determines the appropriate concentration of
fixatives. Concentrations of formalin above 10% tend to cause increased hardening and
shrinkage.
Osmolality of the fixative and ionic composition.
The osmolality of the buffer and the fixative is important, hypertonic and hypotonic
solutions lead to shrinkage and swelling respectively. They can affect the cell shape
and structure. The ionic composition of the solutions should be as isotonic as possible.

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 Chapter Six:

Decalcification:
Learning outcomes
At the end of the instruction the learner should be able to:
a) Define decalcification
b) State the histological importance of decalcification
c) Describe decalcification process
d) Outline the various decalcification methods
e) Describe the characteristics of decalcifying agents
f) Determine the end point of decalcification

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Introduction:
This is the removal of Calcium ions from a tissue to make it soft for easy section
cutting and avoid damage to the microtome knife. Tissues that need decalcification
include:
i) Bone
ii) Teeth
iii) Cartilage
iv) Calcified soft tissues such as Lymphoid and lungs due to Tuberculosis
infection, ovarian cyst and thyroid gland.
Before commencing on decalcification by any method it is essential that the tissue is
thoroughly fixed. It is also essential that the selected tissue should be cut into small
pieces for two reasons.
a) To allow rapid penetration of the fixative and decalcifying fluid.
To reduce time required for decalcification since a prolonged immersion in acid
solution will seriously umpire subsequent staining section.
Methods of decalcification:
The best fixative for tissue to be decalcified is 10% formal saline though others can
also be used.
Methods use for decalcification include
a) Use of mineral acid
b) Ion exchange resin
c) Chelating agents
d) Electrolytic Method.
Qualities of a good decalcification process.
A good decalcifying procedure should ensure that:-
i) There is complete removal of Calcium ions
ii) There is no distortion of tissue structures and cells.
iii) There are no harmful effects on subsequent staining results.
In addition to these criteria the speed of decalcification is important. Use of heat of
370Ccan speed up the decalcification 50 -60 0C an also be used but use of heat is
discouraged the acid digest the tissue.
The volume of the fluid should be 30-50 times the size of the tissue.
a) Use of acids.
The commonest method of decalcification is by dissolving the calcium salt in an acid
medium. There are several acids that are commonly used for decalcification. These
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acids can be mixed just as fixatives are mixed to obtain combined effects, some of
them have fixatives incorporated to continue with fixation process. To avoid digestion
of the tissues by the acids there’re to be divided, most commonly used acids are:-
1. 5-10% nitric acid.
2. 10% formic acid
3. Trichloacetic acid (5%)
4. Perenyi’s fluid.
5. Von –Ebner’s fluid.
6. Formic acid/sodium citrate.
7. Gooding and Stewart’s fluid.
8. Hydrochloric acid

Principle
They react by simply dissolving calcium salt from the tissue, the Calcium ions will
then combine with onions in the solution and stay in a soluble form, if for example,
Hydrochloric acid has been used as a decalcifying agent, Calcium ions will combine
with chloride to form Calcium chloride and hydrogen phosphate.
1. Nitric Acid.
- Used when diluted to 5-10% decalcification time is 2-4 hours recommended for
routine use.
- Rapid in action.
- Causes damage to tissue and inhibit nucleus staining if left in the solution for a
long time.
- Acid develops a yellow solution if stored for a long time due to formation of
nitrous acid this can be avoided by addition of 0.1% urea. Tissue transferred
directly to 70% alcohol
2. Trichloacetic acid.
- Recommended for decalcification of teeth.
- Decalcification time is 2-7 days.
- 5% concentration is the best.
- Tissue transferred directly to 70% alcohol.
3. Formic acid:
- Used at 10% concentration.
- Decalcification time is 2-20 days.
- Used as a decalcification agent in a varied of mixtures.
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 - If higher concentration is used there is cloud formation which prevents
the assessment of the end point.
- No distortion of tissues and no effect on staining sodium citrate can be
added to formal formic acid which gives good results.
- Tissue transferred directly to 70% alcohol.
4. Perenyi’s fluid.
- Composed of:-
- 10% nitric acid -40 ml
- 0.5% chromic acid -30 ml.
- Absolute alcohol -30 ml
- Decalcification time is 2-20 days
- No distortion of tissues and no effect on staining reaction
- No hardening of tissues
- Tissue directly transferred to 70% alcohol.
Disadvantages
- Slow in action
- Chemical test cannot be used to assess the chemical end point ass it
forms a precipitate when ammonium solutions added.
5. Gooding and Stewart’s fluid.
Formula
- Formic acid - 25 millilitres
- 40% formaldehyde -5mls
- Distilled water -100ml
- Addition of formaldehyde projects the tissues against harsh effects e.g.
swelling of tissues.
- Nitric acid can also e added instead of formaldehyde.
6. Von –Ebner’s fluid
Composition
- Saturated aqueous sodium chloride – 50 ml
- Distilled water – 50 ml
- Con. Hydrochloric acid – 8 ml
- Decalcifying time is 3-5 days
- Fairly rapid
- Good for decalcifying of teeth.
- Staining results are good
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 b) Ion exchange Resins:
An ion exchange polystyrene resin like Zeokarb 225 is incorporated at the
bottom of the decalcifying fluid. As the calcium ions are being removed by the
acid (Decalcifying fluid) thus speeding up decalcification. The assessment of
the end point cannot be done by chemical methods due to absence of calcium
ions in the fluid. X- ray method is probably the best.

Container

Fluid

Tissue

Resin

c) Chelating agents:
 These are chemical substances that are able to bind calcium to form
soluble non-ionized compounds examples of this agents are:-
 Ethylene Diamine tetra acetic acid (EDTA).
 Deposits of iron and other metals can also be removed.
 End point is by X –ray and not chemical method due to unavailability
of free calcium ions in the fluid.
EDTA:
 Decalcification time is 4 -40 days.
 Slow acting.
 Tissue aren’t hardened
Composition
i) EDTA – 5.5gm
10% neutral formalin -100ml
ii) EDTA – 250 grams
Distilled water – 1,750ml
Sodium hydroxide -260grams

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 d) Electrolysis:
Calcium ions are removed from tissue by a decalcifying fluid these ions will
then move to the Cathode. The negatively charged ions move to the anode.
This method is not recommended because tissues normally get charred due to
use of electric current, it is also expensive.
End point is determined by physical methods

Anode Cathode

Positive Negative ions

ions

Fluid Tissue

Methods of Determining End point of Decalcification

Method of determining the end points can be divided into
a) Chemical
b) Physical
Chemical Methods
This method is only applicable where mineral acids have been used as a decalcifying
agent
Principle
Calcium ions react with oxalates in an alkaline medium to form calcium oxalate which
is turbid.
Method:
1. Put 5mls of decalcifying fluids into a clean test tube.
2. Put a red litmus paper into the tube.
3. Add strong ammonia drop by drop until the solution is alkaline (paper turns
from re to blue).
4. Add 0.5mls of ammonium oxalate.
5. Read (Observe) if there is no reaction stand it for 30 minutes then read again.
Results:
 Turbidity – Calcium ions present and the tissue should be transferred to fresh
decalcifying fluid.
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  No turbidity – Decalcification is complete and the tissue is moved to the next
stage of processing.
 Note: if Perenyi’s fluid has been used as a decalcifying fluid a modification of
the test is required this is because a precipitate is formed when strong ammonia
solution is added. Dissolve the precipitate that form by adding a few drops of
glacial acetic acid and proceed with the next step.
If turbidity shows after addition of ammonia and the decalcifying fluid is not
Perenyi’s fluid then there is plenty of Calcium ions due to formation of Sodium
hydroxide
Physical Methods.
They can be divided into:
a) Mechanical
b) X-ray
Mechanical method
This involves
i) Probing with fingers to see whether the tissue is still hard
ii) Pricking with a needle to see if it can go through the tissue
iii) Bending the tissue to determine the softness.
Disadvantages
This method is very inaccurate and can lead to distortion of tissues
It’s not possible to detect small amount of calcium
Advantage:
It’s not expensive and easy to carry out.
Radiological Method.
Calcium ions are radio opaque and can be detected through X –rays.
Disadvantages
Not good for tissue fixed in fixatives containing radio opaque substance e.g. mercuric
chloride.
It’s the best method but very expensive
Post Decalcification Procedures:
Depending on the next stage of tissue processing it can be washed thoroughly in water,
transferred to 70% alcohol or stored in 10% formal saline.
Surface decalcification and softening of hard tissues
Difficulties are often encountered in section cutting due to hardness of tissues or
unexpected calcium foci.
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This can often be the major problem in autopsy specimens causing considerable
damage to the microtome knife. These problems are solved by the use of the following
reagents.
1. Perenyi’s fluid.
 Fixed tissues are left in the fluid for 12-24 hours then moved to the next
processing stages or then cut surface of the bloc may be submerged in
the fluid for 1 or 2 hours before cutting.
2. Mollifex:
 Used to soften hard tissue
 Also acts on minute calcium deposits
 Tissue may swell slightly but may not affect subsequent staining.
3. Lendrums
After fixation and washing out the tissue is put in 40% aqueous phenol for 1-3
days or the cut surface of the tissue block is submerged in phenol for this
before cutting.
4. 1% Hydrochloric acid in 70% alcohol.
Can be used to soften the tissues or surface decalcification.

Chapter Seven:

Tissue processing:
Learning outcomes
At the end of the instruction the learner should be able to:
 Define tissue processing.
 Describe all the tissue processing stages.
 Explain the procedure used for selection of tissues for processing.
 Discuss the various characteristics of chemicals and reagents used in the
process.
 Compare automatic and manual tissue processing

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Introduction:
Tissue processing is the treatment of tissue with various reagents to enable production
of thin sections for microscopic examination. There is no perfect tissue processing
technique. There are several techniques utilizing different agents, but all have
advantages and limitations
Tissue processing involves; Dehydration, Clearing, Impregnation and Embedding.
Selection of tissues for Processing.
1. A brief description of the tissue is necessary e.g. nature of tissue, site, if special
attention is required.
 Tissue identification based on the particulars in the requisition form.
 Registration by giving a laboratory number.
2. Small pieces of tissue are trimmed (cut) from the gross specimen.
Factors that affect Tissue processing
Tissues should be small enough so that the processing fluids can easily penetrate in the
time allotted for that step of procedure. Some tissues are more dense ( like the uterus)
while some are less dense ( like the lung), and that it will impact the speed at which the
fluids penetrate the tissue.
Reagents used in processing must have low viscosity, which means that it will
penetrate more rapidly. Examples of high viscosity fluids are honey, molten paraffin
wax and motor fuel while low viscosity fluids include water and alcohol.
Increased temperature speeds up processing steps; however, very high temperatures
can be delirious to the tissue. Heat in alcohol and xylene steps will make tissues more
brittle and dry.
Agitation of tissues when in processing solutions increases the chances of penetration
and as such the tissue should not be stationary during processing.
Vacuum and pressure are available on most modern tissue processors to improve the
removal of air from the tissue spaces and to ensure quick penetration of solutions into
tissue.
In summary factors that have effect on tissue processing comprises of:
1. Size of the tissue
2. Penetrability of tissue (Density)
3. Viscosity of reagents
4. Temperature
5. Agitation
6. Vacuum.
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Dehydration:
-This is the removal of water from tissues, during impregnation with supporting media
like paraffin wax, Celloidin and Low Viscosity nitrocellulose, tissues will not be
impregnated properly if it has H20 since the media and water doesn’t mix.
- Most fixatives used during fixation contain H 2O that is why dehydration is
necessary.
- Dehydration can either be achieved by use of reagents, freeze drying or freeze
substitution.
There are several reagents that can be used for dehydration. They include:
i) Alcohol such as ethyl, methyl, iso-propanol, and Butyl.
ii) Acetone.
iii) Pyridine
iv) Cellulose
v) Dioxane (Diethylene dioxide).
i) Alcohol:
 During dehydration with alcohols the tissue is carried through a sequence
of different concentration starting from the lowest to the highest (ascending
grades) usually used are 30%, 50%,70% and 90% and absolute alcohol ( 3
changes).
 To detect whether dehydration is complete the last bath which is absolute
alcohol, Anhydrous Copper sulphate- wrapped in a filler paper, is placed at
the bottom this acts as an indicator because it turns from white to blue when
in contact with water.
 Dehydration in alcohol takes between 2 – 12 hours depending on the size,
nature of tissue and the temperature at which the process is carried out at
37°C dehydration is enhanced.
 Use of ascending grades of alcohol is recommended because if the tissue is
taken directly to absolute alcohol the tissue will shrink due to abrupt
withdrawal of water.
ii) Acetone
- Dehydration in acetone takes about half an hour. It is a rapid dehydrating
agent and is good for urgent biopsies.
- It causes excessive shrinkage to tissues so tissues.
- Dehydration is done at 4oC on tissue requiring demonstration of enzymes
iii) Dioxane
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 o It is not routinely used because it produces toxic fames.
Advantages
o It mixes both with water and paraffin wax and the tissue doesn’t require
clearing.
o It causes very little shrinkage to tissues
o Dehydration takes 3 -24 hours depending on the type and size of tissues
Disadvantages
o It is a very expensive reagent as compared to other dehydrating
solutions.
Freeze drying
This is also known as lyophilization or cryodesication, it is a dehydration process
typically used to preserve a perishable material or make the material more convenient
for transport.
Freeze drying works by freezing the material and then reducing the surrounding
pressure and adding enough heat to allow the frozen water in the material to sublime
directly from solid phase to gas.
The process is often done by placing the material in a freeze drying flask or chamber
and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical
refrigeration, dry ice and methanol or liquid nitrogen.
On a larger scale freezing is usually done using a freeze-drying machine. Normally the
freezing temperatures are between -50oC and -80oC.
The freezing phase in the whole dry-freezing process is done carefully because the
product can be spoilt if badly done.
The pressure is then lowered and enough heat is supplied to the material for the water
to sublimate. The amount of heat necessary can be calculated using the sublimating
molecules ‘latent heat of sublimation’. The pressure is controlled through the
application of partial vacuum. The vacuum speeds sublimation, making useful as a
deliberate drying process.
Freeze drying causes less damage to the substance than other dehydration methods
using higher temperatures. It does not usually cause shrinkage or toughening of the
material being dried. However, water is not the only chemical capable of sublimation,
and the loss of other volatile compounds can yield undesirable results.
Freeze drying can also be used in food preservation, pharmaceutical and
biotechnological industries.

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Freeze Substitution
This is a modification of freeze drying. Ice within the frozen tissue is replaced with
alcohol or any other solution at low temperature, dry acetone can be used as a
substitute.
Freeze substitution involves the replacement of ice in a frozen specimen with an
organic solvent at a higher temperature than that at which the specimen was frozen.
Fixation continues to occur at a low temperature during the substitution process
resulting in less extraction. The solvent is then replaced by a resin for specimen
embedding.
In general, ultra-structure preservation is improved by slow dehydration and the low
temperature embedding. Epoxy resin can be used for embedding.
The substitution is usually carried out at -180oC to -95oC and can be performed on
either aldehyde fixed specimen or on rapidly frozen (unfixed material). The material
can then be sectioned for morphology examination.
Clearing
- This is the removal of alcohols or any other dehydrating agent from tissues.
- It is done because most of the dehydrating agents do not mix with the impregnating
or supporting media such as paraffin wax.
- The first clearing agents that were used rendered tissues transparent and this effect
led to the use of the word “Clearing”.
- Not all clearing agents used at the moment render tissues transparent clearing agents
Commonly use clearing include: - Xylene, Toluene, Chloroform, Cedar wood oil,
Paraffin oil and Aniline oil.
a) Xylene
Advantages:
o Tissue are made transparent
o Rapid in action taking 2-6 hours
o Not expensive compared to other clearing agents.
o Easily removed form tissues during impregnation with supporting
media.
o No effects on subsequent tissue staining.
Disadvantages:
o Tissue become brittle on long immersion in xylene
o It causes excessive shrinkage on delicate tissues.

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 o It is highly flammable
o Digest all plastics and rubbers so can’t be kept in such containers.
b) Chloroform
Advantages:
o Non flammable
o No much shrinkage to tissue
o Tissues don’t become brittle on long immersion
o No effect on staining reactions.
Disadvantages
o 3 -5 times more expensive than Xylene.
o It is slow in action
o Doesn’t make tissues transparent
o Its vapour is anaesthetic and toxic
o Tissues float on top due to its high S –G.
o Not easily removed from tissues during impregnation.
c) Toluene
It clears tissues up to 2 mm in 2 hours
Advantages:
o It doesn’t make tissues brittle.
o It is a rapid clearing agent
o Tissue become transparent
Disadvantages
o Have toxic fumes.
o Has a tendency to become acidic.
d) Benzene
Advantages
o It fairly rapid in action (can clear 3mm in 3 hours).
o Doesn’t make tissue brittle
o Causes little shrinkage
o Is easily eliminated from paraffin wax.
Disadvantages
o Highly flammable
o Has toxic fumes

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 o Is carcinogenic especially if used for long times (cancer causing
reagents).
e) Cedar wood oil
Advantages
o Cause little or no shrinkage even if the tissue is left indefinitely.
o Tissues become transparent
o No hardening of tissues.
Disadvantages:
o It is a very expensive agent as compared to other clearing agents.
o It is slow in action both in replacement of alcohol and being replaced
during impregnation.
o It sometimes partially solidifies through formation of needle like
crystals.
Impregnation:
This is also known as infiltration and can be defined as the saturation of the tissue with
the infiltration medium. It is done to remove the clearing agent and air form tissues
and also to allow the impregnating media penetrate into the tissue so that a block is
produced while be cut into sections (slices).
Wax is the commonest media used for impregnation. Waxes mostly used for this
purposes include: - Paraffin wax, Ester wax and Water soluble waxes.
Other impregnating media that can be used instead f waxes are; Gelatine, Agar, Low
viscosity nitrocellulose (LVN) and Celloidin.
i) Impregnation with paraffin wax:
Paraffin wax.
o It is a mixture of hydrocarbons produced during the cooking of mineral
oil the melting point ranges between 40ºc-70ºc.
o It contains additives which harden the wax to facilitate production of
these sections.
o Common additives include :-
i. Bee wax
ii. Ceresin
iii. Diethylene glycol distearate
Other forms of paraffin wax used are:
- Para-blast, Para-blast plus, bioloid and tissue mat.

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Infiltration is done using ordinary hot air oven or a vacuum chamber. In both cases the
P. wax is kept molten at 2 -3° C above the melting point of wax.
Hot air oven:
Tissues are transferred from the clearing agent to a bath of molten paraffin
- The underlying principle is that the clearing agent and air are removed from the
tissue.
- The paraffin wax in turn diffuses or infiltrates into the tissues to replace them.
Because of the high temperature the clearing agent being volatile evaporates.
- Two changes of wax bath are necessary for complete impregnation (about 2 hours in
each).
Vacuum impregnation:
- This is done in a chamber which operates under reduced pressure.
- It enables the clearing agent and air to be rapidly removed from the tissues thus
allowing quick penetration of the wax into the tissues. This reduces the time to 30
minutes.
- Two wax baths are necessary for complete impregnation (15 minutes in each bath).
- The method is not routinely used because the equipment is expensive and the space
is small.
This method is mostly recommended for the following specimens.
 Urgent Biopsies
 All decalcified tissues like bone
 Fatty tissues
 Tissues containing Blood clots such as. Liver, spleen.
 Tissues with a lot of air spaces an example is the lung tissue
 All specimens form central nervous system
 Tissues made of delicate structures and of different consistency like. the eye.
Method of use
i) Put tissue form the clearing agent into molten paraffin wax at 2 -3 oC above
melting point of wax.
ii) Place it in the chamber.
iii) Close the chamber using a heavily lid
iv) Make the chamber air right
v) Exhaust air slowly by vacuum or suction pump till the manometer reads
400-500 mmHg
vi) Maintain the pressure for 15 minutes
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 vii) Read mix the air slowly till normal atmospheric pressure is attained.
viii) Repeat once more using fresh paraffin wax
ix) Bring to normal atmospheric pressure.
x) Remove the tissue and embed.
xi) The temperature of the chamber is controlled by a thermostat.
xii) There are several types of vacuum impregnating chambers. Some have a
single chamber others have double.
xiii) They differ from one manufacturer to another but they have the same
working principle.

Vacuum impregnation chamber

Lid

Thermometer
Manometer

Chamber
Sanction pump

Paraffin wax
Tissue
Water
Jacket
Heating element

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Embedding:
This is also known as blocking out or casting. It is a stage whereby tissue is supported
by paraffin wax or any other medium by being made into a block ready for section
cutting.
- To obtain a block various objects like, moulds are used to hold the paraffin wax
and tissue so that on solidifying become a block.
- The moulds commonly used are:-
o Leuckharts (L-shaped moulds).
o Paper boats.
o Tissue Tek
o Plastic ice cubes
o Aluminium coils
o Wooden boxes.
o Petri dishes.
Different people have applied different techniques to prepare blocks. An example is
outlined.
- Put the mould in a suitable plane.
- Fill it with molten paraffin wax.
- Warm a pair of forceps and use it to transfer the tissue from the last bath of wax.
-Place the tissue in the desired plane or the surface to be cut facing down.
- Let it cool until it solidifies. (Water and ice can be used to enhance solidification).
- Trim to remove excess wax thus exposing the surface of the tissue.
- The blocks are durable when kept in cool place.
Advantages of paraffin wax.
- Fairly rapid in action as compared to other impregnation media
- Provide good consistency of serial section cutting.
- Give durable blocks which provide no storage problems.
- It is cheap as compared to other embedding medium
Disadvantages.
- Hardens and shrinks the tissue.
- Bones are difficult to cut even if they have been decalcified
- Thick sections of blood specimen are difficult to cut.
- Soft tissues e.g. liver and spleen crack during section cutting.

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- Tissue structures tend to get distorted due to application of heat.
ii) Impregnation with water soluble waxes
Commonly use water soluble waxes are polyethylene glycol 1000 and polyethylene
glycol 1500. They are graded according to molecular weights. The melting point
ranges from 38°C - 42°C
Advantages of water soluble waxes:
It causes little shrinkage.
No need for dehydration and clearing
Tissues are not exposed to high temperature.
These waxes are useful when demonstrating tissue structures that are sensitive to heat
like enzymes, lipids, antigens and antibodies.
Disadvantages:
Sections are difficult to float on the water bath and mounting on the microscope slide
Blocks must be kept in dry atmosphere as it will dissolve in humid environment.
They do not penetrate tissues readily or fast.
Techniques:
Fix the tissue and wash in water
- Impregnate in 70% polyethylene glycol in water for 45.minutes
-Impregnate in 90% polyethylene glycol in water for I hr.
- Impregnate in 3 changes of 100% polyethylene glycol for 25 minutes in each.
- Embed in 100% polyethylene glycol cool blocks in the refrigerator at 4ºC
- Cut sections.
iii) Impregnation with Ester waxes
Ester wax is a mixture of diethylene glycol distearate, glycol monostearate and 300-
polyethylene glycol. It is for impregnation of bone tissue and insects.
Advantages
- It has a low Melting Point. of 45° C to 47º C
- Gives hard blocks which can allow cutting of 10 microns thick sections.
- Produces good ribbons
- It is soluble in diethyl alcohol.
- No clearing is required because cellusolve mixes with the wax
Disadvantages
- It is 8 times more expensive than paraffin wax.
Technique
- Dehydrate tissues in 70% cellusolve for 8 hours.
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- Dehydrate tissues in 90% cellusolve overnight 12 – 18 hours.
- Dehydrate in pure cellusolve 3 changes for 2 ½ hours in each.
- Impregnate in equal parts of cellusolve and ester wax at 37° C overnight.
- Impregnate the tissue with 3 changes of ester wax for I hr.
- Impregnate the tissue with ester wax in vacuum bath for 10-45 minutes.
- Embed in freshly melted ester wax.
- Cool in a refrigerator and cut sections using a heavy duty microtome such as sliding
and base sledge.
- Dry the section at 370C because they crumble if dried at room temp.
iv) Impregnation with Celloidin.
- Celloidin is purified form of Low Viscosity Nitrocellulose (LVN) it is best for
impregnation of hard tissues and those of mixed consistency. It allows thick sections
to be cut. There is minimum shrinkage and distortion of tissue since no heat is applied.
- The working strength is 2% (thin), 4% (medium) and 8% (thick) the solvent being
equal volume of ether and ethanol.
It is usually supplied as shreds or wool moistened with alcohol.
Advantages:
- Gives much support so that large tissues can be processed and embedded.
- Tissues with different layers are impregnated without affecting the relationship of the
various layers
- Useful for hard or fragile tissue such as brain and heart
- Does not require heat so no distortion of tissues
- It causes minimum shrinkage and hardening of tissue.
- Miscible with dehydrating agent so no clearing is required.
Disadvantages:
- Slow taking several days
- Only thick sections can be cut 10-20 microns
- Blocks must be stored in 70% ethanol
- It is highly flammable and open flames should be avoided at all costs
- Difficult to produce serial sections.
- Blocks and knives must be continuously moistened with 70% ethanol during cutting.
Technique:
- After dehydrating place the issue in ether for 12-24 hours.
- Place the tissue in 2% Celloidin 5-7 days
- Place the tissue in 4% colloidal 5-7 days
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- Place the tissue in 8% colloidal 5-7 days
- Embed in thick Celloidin 8% and let it harden
- Store the block in 70% alcohol till ready for cutting.
v) Impregnation with Low Viscosity Nitrocellulose (LVN)
-Has low melting point than Celloidin
- Penetrates more readily than Celloidin
- Sections tend to crack when embedded in LVN.
- A plasticizer such as tricrsyl phosphate, castor oil or oleium resin can be incorporated
to overcome the problem.
- Blocks formed are harder than those of Celloidin
- The technique of processing is the same as that of celloidin except the concentration
applied is different (10%, 20% and 50%).
vi). Impregnation with Gelatin
- Used to embed fragile tissues such as Curettin (scrapping from endometrial wall) and
cystic structures
- Used as embedding media when cutting frozen sections for demonstration of delicate
structures that can be affected by reagents and heat
- Impregnation is carried out at 37 degrees

vi). Synthetic resin media

They are media that allow cutting of very thin tissue sections from 0.005-0.01 microns.
The type of knife used is made of glass on an ultra- microtome. The sections are
examined using an electron microscope.
It is used to embed exceptionally hard tissues like undecalcified bones. Plasticizers are
added into the media to improve on elasticity.
The media include acrylic resins such as araldite and epoxy resins like methacrylate.
Pertefi’s double embedding media
- Involves use of paraffin wax and Celloidin.
- Impregnation is first done by the use of paraffin wax then embedded in the same
- It is useful for the tissues with mixed consistency, hard tissues like eye, brain, muscle
fibroids and uterus.
Technique:
- Dehydrate tissue normally
-Transfer tissue to methyl benzoate/Celloidin mixture for 24 – 72 hours.
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- Pass tissue through 3 changes of benzene or toluene for 24 hours.
- Impregnate and embed in Paraffin Wax.
- The method is not rapid but is advantageous in that it produces improved cohesion of
tissue layers and plasticity given by Celloidin with the facility of producing ribbons
which is characteristic of paraffin wax.
Schedules of Paraffin wax processing
Processing using Paraffin Wax can be done manually or by use of automatic tissue
processor.
- Rapid Tissue Processing- Takes a maximum of 4 hours
- Routine processing takes 22 – 48 hours
Manual processing
The time schedule is determined by the size, nature of tissue and the type of solution to
be used. Paraffin wax processing of small tissues of less than 2 mm is as follows:
1. 70% ethanol -20 minutes
2. 95% ethanol -20 minutes
3. Absolute ethanol I – 30minutes
4. Absolute ethanol II – 20minutes
5. Xylene I – 30 minutes
6. Xylene II – 30 minutes
7. Wax bath vacuum I – 1hour
8. Wax bath vacuum II -1 hour
Total – 4 ½ hours
This is followed by embedding in Paraffin Wax
Rapid manual processing of tissues of 2 mm thick can be carried out as follows:-
1. Slice 2 mm thick pieces of tissue
2. Boil in 10% formal saline – 5 min
3. Cool to about 500C
4. Dehydrate in 70% ethanol - 10 minutes at 600 C
5. Dehydrate in 95% ethanol -10 minutes at 60 0C
6. Dehydrate in absolute ethanol I - 10 minutes at 600 C
7. Dehydrate in absolute ethanol II - 10 minutes at 600 C
8. Clear in xylene I for 15 minutes in vacuum chamber
9. Clear in xylene II for 15 minutes in vacuum chamber
10. Impregnate for 15 minutes in Paraffin Wax in Vacuum Chamber.
11. Impregnate for 15 minutes in Paraffin wax in a vacuum chamber
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 12. Impregnate for 20 minutes in wax bath III
13. Embed in Paraffin Wax cool in the refrigerator and cut sections as required
Automatic Tissue Processor:
There are different types of tissues processors but they have the same functioning
principles
Examples are
a) Histokinette
b) Tissue tek II
c) Autotechniron Duo
d) Histotechnicon ultra
e) Tissue maton
f) Histomatic model 165
The advantages of tissue processors are;
1. Time required in each reagent is reduced due to
a. Suspending tissue in fluid
b. Continuous agitation
c. Movement of tissue from one reagent to the other in a desirable manner
and not controlled by normal working hours

2. Free laboratory staff for work of more technical nature

General construction of a tissue processor.

It consists of the following:-
 Body
 Transfer arm
 Beakers and wax baths
 Agitation mechanism
 Delay mechanism
 Safety devices
 Tissue containers
 Timing mechanism
Body: It is made up of resin fibre glass. The body is impervious to all reagents
used during processing and is mounted on a roller so that the platform turns and
makes it easy to reach the beakers.
Transfer arm: - The arm moves the tissues according to a predetermined time.
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 Beakers and wax baths: There are 10 beakers and 2 wax baths which are
thermostatically controlled. The beakers contain the processing
reagents/solutions. Wax bath contains molten paraffin wax at 2-30C above
melting point of wax.
Agitation- There is a system which provides continuous vertical movement
through the transfer arm which the tissue baskets are connected. The system is
responsible for moving the tissue up and down so that there is faster penetration
of the solutions.
Timing mechanism. It consists of an elastic clock that is connected to a disc
marked to cover a period of 24 hours.
Delay mechanism: The mechanism permits the machine to work over the
weekend and tissues are ready for embedding on Monday morning.
Safety devices: - These are the cut off safety devices that warn in case of a
problem through an alarm or an indicator light. They enable the tissue to remain
in wax bath after the end of the processing time and to act as an emergency
incase of wax solidifying in one container.
Tissue containers: The mostly used containers are;
 Plastic tissue basket
 Metal baskets
 Curettin baskets.

Schedule for Automatic tissue processing

The processing of tissues using an automatic tissue processor is set according to the
solution/reagent to be used. The following are examples of set schedule
Schedule I Schedule II
70% ethanol - 1 hr 70% ethanol - 3 hr
96% ethanol I - 1 hr 90% ethanol I - 3 hr
96% ethanol II - 2 hrs Absolute ethanol I - 1 hour
Absolute ethanol I - 1 hr Absolute Ethanol II - I hour
Absolute Ethanol II - 2 hrs Absolute Ethanol III - 2 hours
Absolute Ethanol III - 2 hrs Absolute Ethanol chloroform IV – 2 hours
Absolute ethanol chloroform – 1 hour Toluene I – I ½ hours
Chloroform I – I ½ hours Toluene II - 2 ½ hours
Chloroform II - 2 ½ hours Toluene II – 3 hours

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Chloroform II – 3 hours
Chloroform III – 3hours
Paraffin Wax I – 2 hours
Paraffin Wax II – 3 hours
Total - 22 hours Total - 22 hours

Chapter Eight:

Microtomy (Section Cutting)

Learning outcomes
At the end of instruction the leaner should be able to:
 Define Microtomy
 State the use of a microtome
 Classify microtomes
 Describe the basic construction of a microtome
 State the use of the various knife profiles
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  Describe knife sharpening both manually and automatically
 Discuss tissue section cutting
Introduction
Microtomy is the cutting of thin tissue sections using special machines called
microtomes for microscopic examination.
The thickness of the sections depends on the type of tissue and the embedding media
used; routinely 5 – 10 microns are preferred.
Microtomes are defined as instruments used to cut accurately measured thin sections
that will allow light to pass through during microscopic examination.
There are varieties of microtomes used depending on;
a) Type of work such as demonstration of fat and enzymes
b) Nature of tissue preparation required
c) Embedding medium to be used
Microtomes:
Some of the commonly used microtomes include:-
- Rocking (Cambridge rocker)
- Rotary
- Sliding
- Base sledge
- Freezing
- Cryostat
- Ultra
- Thermo- electric device
Though they differ in their construction they have some common features, these are:-
 They have a knife, held in a firm support
 Have a device for holding a knife
 Have a device for holding the tissue block
 Have a mechanism for moving the tissue block across the knife or knife across
the tissue
 Have mechanisms for advancing the pieces of tissue through very short and
accurately measured distance so as to obtain a tissue section of suitable
thickness.
Rocking microtome
- Designed for cutting Paraffin Wax tissues blocks.
- Suitable for soft small tissues
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- Not suitable for large blocks of tissues and also hard tissues like bones because it is
light in weight
- It is not recommended for serial sections because it produces curved sections.
- The knife is fixed with the edge uppermost while the object moves against the knife
in an arc of circle producing a slightly curved surface block.
- It is relatively cheap as compared to other microtomes.
- Its knife is also cheap in terms of cost and does not require a knife back when
sharpening like those of other microtomes.
Rotary Microtome
Basic Construction:
- This is a heavy microtome with the feed mechanism encased to avoid dust and dirt
- The handle, knife adjustments, block, block holder and thickness gauge are extremely
exposed.
- Block holder has an adjustment attached to the feed mechanism and it protrudes
outside towards the knife adjustment.
- The feed mechanism consists of pawl, ratchet wheel and micrometer screw.
- The micrometer screw is mounted on the ratchet wheel; the ratchet wheel is
connected to the block holder by a clip nut.

Operation:
- By running the microtome handle through one revolution it causes the pawl to engage
the ratchet wheel which turns the micrometer screw through a distance that is pre-
determined by the thickness gauge
- Rotary microtome can be used for cutting large blocks of paraffin wax embedded
tissues
- The block holder moves up and down vertically in front of the knife cutting sections
in the process.
- It is good for cutting of serial sections therefore is recommended for routine and
research histological work.
Sliding Microtome:
- It is recommended for cutting of both Paraffin Wax and celloidin embedded tissue
blocks.
- The tissue block remains stationary while the knife moves forwards and backwards
along sledge that is fixed to a heavy metal base during the process of sectioning.
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- Mostly used for cutting nitrocellulose embedded tissues
Base sledge microtome
- It is designed for cutting both paraffin and celloidin embedded tissue blocks
- The tissue block is attached onto a sliding base that moves to and fro, the block
comes across the stationary knife thus producing sections.
- It is suitable for cellulose nitrate embedded tissue blocks.
-Good for hard and large specimens particularly bone and teeth.
-When the knife is set obliquely it is used to cut LVN blocks and when set at right
angle to the direction of the block can be used for paraffin embedded blocks.
Freezing microtome
- It is a light microtome with working parts externally exposed. The handle, the knife
holder, block holder and thickness gauge are also exposed. The block holder has an
adjustment attached to the feed mechanism.
- The feed mechanism consists of the pawl, ratchet wheel and micrometer screw.
- The micrometer screw is mounted onto a ratchet wheel. In some freezing
microtomes the block holder is serrated to hold the tissue firmly onto it when freezing
takes place, others are hollow. Freezing microtome use compressed CO 2 or liquid
nitrogen to freeze the tissue.
Operation:
- By turning the microtome handle it turns the ratchet wheel.
- The knife is drawn towards the operator against the vertical holder
- The knife pull cause the pawl to engage the ratchet wheel which turns the micrometer
screw through an arc of a cycle through a pre- determined distance set by thickness
gauge.
- The knife moves while the tissue block is stationary.
Application of freezing microtome:
1. During demonstration of fat.
2. For demonstration of histochemcial tissue structures such enzymes
3. Urgent examination
4. Neurological studies
Disadvantages:
1. Production of serial sections and ribbon is difficult.
2. Sections are cut at an uneven thickness.
Cryostat:

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- It consists of a microtome contained within a refrigerated cabinet designed to operate
at -50 C to -300 C. Any rotary microtome can be modified for use in the cryostat by
using stainless steel components.
- Cambridge rocker microtome is widely used because it has few moving parts
- The optimum cutting temperature is -200C.
- It is used to cut fresh, frozen, unfixed tissues for demonstration of structures like
antibodies, fats, enzymes.
- The knife and tissue must be at the same temperature and the tissues must be kept in
the microtome chamber in an air tight container at -20 0C if they are not to be sectioned
immediately.
Note: The application of a cryostat on tissue sections are the same as those of freezing
microtome even on the disadvantages.
Thermo electric device
- Thermo electric cooling unit may be use in place of CO 2 to freeze tissues and cool the
knife
- When a direct current is passed across the function of dissimilar metals, heat is
emitted or absorbed depending on the direction of the current
- A flow of cold H2O maintained through the cooling unit ensure that the heat from the
hot face (tissue and Knife) is absorbed
- The optimum cutting temperature is -200C.

Ultra microtome
- Are designed for preparation of very thin sections for examination with electron
microscope.
- They differ with other microtomes in that:
o Fineness and precision of feed mechanism which is either activated
mechanically or electrically to give a thickness of 0.005- 0.1microns
o Use specially prepared knives made of glass plates
o Cuts small blocks of tissues embedded in synthetic resins such as
araldite.
o Care and maintenance of microtomes:
 Remove the knife from the microtome after use
 Clean the microtome with a piece of cloth soaked in Xylene after use
 Clean the wax/tissue pieces with a camel hair brush after use.
 Cover the microtome with a plastic bag and keep in a dust free room
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  Keep microtomes on a stable bench to avoid vibrations
Maintenance:
a) Occasionally lubricate the moving parts with oil
b) Tighten the screws and nuts when loose
c) Inspect for damage regularly and consult a repair technician regularly
d) Service the machine regularly
Microtome knives:
- Microtome knives used for cutting of tissue sections are very sharp. They are
made of steel, diamond or glass depending on the manufactures’ wish.
- The knives are available in various types and sizes according to the type of
microtome and embedding media used to make the block of tissue.
- Microtome knives are classified according to the way in which they are ground
or sharpened each meant for a specific purpose.
1. Plano Concave:
- One side is hollow while the other is flat.
- It is mainly used for sectioning nitrocellulose (Celloidin and LVN) embedded
blocks
- It is mostly used to cut sections on a sliding microtome
2. Semi- Plano concave
- One side is slightly concave.
- It is used for cutting of sections from paraffin wax embedded blocks.
- Used in rotary, rocker or base sledge microtomes.

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3. Biconcave
- It is hollow and can be ground on both sides
- Mainly use for cutting Paraffin Wax embedded blocks.
- Mainly used on rotary and Cambridge rocker microtomes. But can be used in
any type of microtome to cut blocks embedded in Paraffin Wax, cellulose
nitrate, synthetic resin and frozen sections.
4. Plane wedge
- It is flat on both sides
- It has a wide range of applications
- Mainly used in any type of microtome to cut blocks embedded in Paraffin Wax,
cellulose nitrate, synthetic resin and frozen section
5. Tool edge (chisel shaped)
- Used to cut exceptionally hard tissues like undecalcified bones used in base
sledge microtome.
6. Glass knife
- Used to cut tissue blocks embedded in synthetic resin such as araldite using ultra
microtome.
Care of microtome
1. Knives should be stored in their boxes when not in use
2. Knives should never be laid flat on the bench
3. Never allow edge of knife to be badly kicked
4. Knife edge should be touched up (sharpen ) daily before use

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5. Always use a knife back. When sharpening Plano concave and plane wedge knife
6. Knives should be cleaned with xylene before and after use
7. Knives should no be allowed to rust
8. Never lend or borrow a microtome knife to avoid misuse
Knife sharpening
- A blunt or damaged knife edge will cause many faults in sections or will fail to cut
any sections at all.
- Bluntness due to the edge of the knife becoming rounded and damaged. It
takes the form of large or small kicks, teeth or striations caused by the breaking
of metal edge.
- Knife sharpening consists of;
i. Honing
ii. Stropping
Honing
Honing is the sharpening of a knife using a hone (stone) to give a sharp cutting
edge to the knife. It is very necessary that the cutting edge is not blunt or nicked if
correct sections are to be obtained.
There are several types of hones for sharpening the knives
Sharpening can be done manually or using an automatic knife sharpener.
The commonly used hones include
 Belgium – yellow stone
 Arkansas stone
 Fine Carborandum
 Glass plate (made of glass not stone).
1. Belgium Yellow stone
Is a natural stone and is best for manual honing
2. Arkansas stone
It is an artificial stone with more polishing effect than the Belgium yellow stone.
3. Fine Carborandum
Fine Carborandum is an artificial stone with a much coarser bite than either the,
Belgium yellow or Arkansas stones. It should only be used for badly kicked knife.
4. Glass plate
It is a piece of glass measuring 8” x 3” x 1”
Abrasives (Lubricants) for hones

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 - Before a hone is used the surface must be lubricated by addition of an abrasive
to allow a smooth movement of the knife during sharpening.
- Any of the following can be used
 Soapy water
 3 in I oil (Mixture of light oils of animals and vegetables).
 Mineral oil USP (liquid paraffin) castor oil, Glove oil, Xylene etc
 Aluminium oxide
- Honing is the process of grinding the cutting edge of a knife to acquire an even
edge. The cutting edge of a knife is called a bevel.
- During sharpening the knife is laid flat on the hone and pushed up the hone, the
cutting leading the stroke in a heel to toe movement (The heel of the knife is
the part nearest the handle).
- On reaching the end of the hone, the knife is turned on its back never leaving
the hone and draws steadily towards the operator. The process may be repeated
10 – 20 times for a knife in good condition but more for a very blunt knife.
- Plane wedge and Plano concave knives require a false knife back when
sharpening so that the cutting edge came in contact with the hone.
- Coarse abrasive is used follow by the fine abrasive to give a polishing effect
- No extra force is used during honing.
Stropping:
Stropping is the refining of the knife edge by using horse hide (skin), any leather or
fine Carborandum
Purpose
To polish the cutting edge and to remove iron fillings (buzz)
Types of strops
There are various types but they are all made of canvas and leather. The leather
used must be of good quality.
Most strops have one side being canvas and the other leather so during stropping
start with canvas followed by leather
When stropping the knife it is laid flat on the strop. The back side of the knife
leads the stroke, on reaching the end it is turned on its back and never leaving the
strop.

Knife sharpening machines

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 - These machines can be semi -automatic or fully automated. They contain the
following
i. Body
ii. Switch
iii. Speed control
iv. Safety device
v. Knife turning mechanism
vi. Glass or metal plate
vii. Knife holder
viii. Knife or plate moving mechanism
 The machines are time saving but are expensive
 They provide uniform performance of the knife cutting edges
 Some machines have both honing and stropping facilities incorporated in them.
Usually the plate (Glass/ metal) rotate and this facilitates knife sharpening.
 The knife is usually turned at certain intervals so that both side are sharpened
equally
 Abrasives used on the plate include aluminium oxide and diamond grit
depending on the condition of the knife.
 The choice of speed is provided by the condition of the knife a slow speed is
required for poor knives
Section cutting of paraffin wax block
1. Fix the tissue onto the tissue block
2. Insert a suitable knife to the microtome block holder and secure it with
tightening nuts or screws
3. Move the block holder with the Paraffin wax block to almost touching the knife
edge
4. Check and tighten all the screws to avoid movement of knife or block during
cutting.
5. The gauge controlling the sections is set between 5 and 20 microns
6. The extreme end of the knife is used to trim the block until full surface is being
cut.
7. Set the required thickness and position the knife so that the centre of the knife
blade is positioned for cutting.
8. Operate the microtome until complete sections are being cut and maintain a
regular cutting rhythm.
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  The cutting rate varies with the nature of the tissue ,the size of the block
and the type of microtome
 If the block faces upper and lower edges of the knife the sections will form
a ribbon. The ribboning is due to the slight heat being generated between
the knife edge and the block
9. Pick cut sections using camel hair brush and put on a dry crease free slide, the
section should be flat on the slide
10. Add few drops of 20% alcohol or 1% acid alcohol beneath the section to flatten
the sections.
11. The section is then lowered slightly onto the surface of warm water in a water
bath set at 6 -100C below the melting point of wax. This process is known as
floating and is to flatten and expand the section so that creases are removed.
12. A prepared clean and grease free glass slide smeared with an adhesive then is
dipped obliquely into the water as close to the section as possible
13. Slowly withdraw the slide allowing its surface to touch the edge of the section,
this process is called fishing.
14. Completely remove the slide with attached section from the water. Adjust the
section to suitable position (at the centre of the slide) on slide using mounting
needle.
15. Drain off the water on a slide rack and transfer it to an incubator or a hot plate
for at least I hour this is to remove the wax. Leaving the section on the slide
16. The temperature of the incubator or the hot plat is 2 – 3 0C above the melting
point of wax.
Section adhesive
These are substances that are used between the tissue section and the slide to stick the
section firmly onto the slide. When section is put in the incubator or the hot plate the
proteinous adhesive coagulate thus making the section stick firmly on the slide
During staining of sections they can partly or completely detached from the slides due
to
 Prolonged immersion of sections in alkaline solutions such as ammonical
silver solutions used during metallic impregnation
 Fixing of tissue in powerful protein coagulant fluids like Bouin’s fluid
 Tissue containing blood clots or bone may also get detached during staining
 Tissue with a lot of air spaces can be lifted from the slide by the staining
solutions.
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The detachment of the tissue section can be avoided by use of section adhesives,
mostly used adhesive are
Mayer’s Egg albumin
- Combine equal parts of glycerol and the white of an egg and filter
- Add small crystal of thymol to act as preservative. Fresh serum,
albumin or any other protein can be used instead of the egg.
Starch paste
Powder starch – 3 g
Cold distilled H2O– 30mls (solvent).
- Mix to make a paste and add to boiling distilled H 2O – 60 ml (provide acid
medium ) Concentrated hydrochloric acid – 0.5mls
- Boil for 5 minutes cool and add thymol crystal to act as preservative
Difficulties encountered in section cutting:
During section cutting good sections may not obtained due to:-
Inadequate impregnation
- Improper dehydration, clearing or wax impregnation will result in section crumbling
- This problem can be overcome by melting the wax and taking the tissue for
reprocessing
Imperfect knife edge
- Nicked knife cut sections with vertical lines that are seen in sections. These lines are
known as scores
- Blunt knife causes alternating thin and thick sections to be cut.
- Crumbling of sections can also occur if a blunt knife is used
- The problems can be solved by sharpening the knife
In- correct setting of the knife
- During section cutting the knife is set at an angle called angle of tilt on the
microtome to allow a clearance angle between the cutting facet of the knife and the
block of tissue. The angle is determined by the type of knife
- Faults that occur due to incorrect results include;
Chatters:
- Chatters are horizontal lines or furrows across the section. This is caused by large
(great) angle of tilt between the knife facet and block of tissue. The problem may be
solved by reducing the tilt angle.
Intermittent cutting:

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- Small angle of tilt allow the block to be compressed, the compression increases until
the tissue expands suddenly and the result is the cutting of thick sections. This can be
avoided by increasing the angle of tilt.
Dirt:
- Presence of dirt in the embedding media and the knife edge cause scores, to avoid the
fault clean embedding media and knife edge must be used.
Minute particles of calcium:
- Tissues containing minute calcium ions foci will break into small pieces when
cut .To avoid the breakage decalcification of tissues must be done to completion before
tissue processing commences.
- Hard tissues break into small pieces during cutting and must be made soft before
sectioning by use of 4% phenol. Surface decalcification can be done using 1% acid
alcohol in order to cut uniform sections.
Faults encountered during section cutting (Summary):
Fault Cause Remedy
1 Tissue  Static electricity
sections cling  Knife edge dirty  Use clean knife
to the knife  Knife edge dull/blunt  Sharpen the knife
 Knife tilt too vertical  Increase angle of tilt
2 Varying  Blocks too large  Use smaller blocks
thickness of  Blocks too hard  Use a softer embedding
sections  Microtome not adjusted media
correctly  Adjust the microtome
 Loose screw on knife setting correctly
or block holder  Tighten all screws
3 Failure of  Block not parallel to  Knife should be parallel
blocks to knife edge to the block
ribbon  Dull knife edge  Sharpen the knife
 Paraffin too hard  Use soft paraffin wax
 Knife tilt too much  Reduce tilt of the knife
 Sections too thick
4 Split sections  Nicks on knife edge  Sharpen the knife
(scores)  Knife edge dirty  Clean the knife
 Too much knife tilt  Reduce angle of tilt
 Calcium foci, foreign  Decalcify tissue
materials on wax or properly, use purified
tissue wax, remove dirt or
crystals from tissue
5 Compressed,  Knife dull  Sharpen the knife
wrinkled or  Paraffin wax too warm  Cool the block to
jammed  Paraffin wax on knife solidify properly
sections  Sections too thin  Clean the knife
 Microtome screws too  Tighten all microtome
loose screws
6 Uneven and  Irregularly trimmed  Trim the blocks

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 cracked blocks uniformly
sections  Edge of block not  Knife and block should
parallel to knife edge be parallel
 Irregularity of in knife  Knife sharpening
edge should be done
 Paraffin wax not uniformly
homogeneous  Use one type of
paraffin wax
7 Tearing and  Incomplete fixation,  Complete fixation,
crumbling of dehydration or clearing dehydration or clearing
sections of tissues  Complete impregnation
 Incomplete with wax
impregnation with  Use soft wax or cool
paraffin wax the block in ice
 Paraffin wax hot or soft
8 Thick and  Tissue too hard  Surface decalcification
thin zones in  Excessive tilt of the or softening of tissue
each section knife  Reduce angle of tilt of
(chatters)  Movement of the knife
microtome during  Put the microtome on a
cutting firm bench
9 Lifting of  Too vertical knife tilt  Set proper tilt of the
sections from  Dull knife edge knife
knife on  Too soft paraffin wax  Sharpen the knife
upstroke  Cool the block in ice

Chapter Nine:

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Staining:
Learning Outcomes
At the end of the instruction the learner should be able to:
 Define staining
 State the importance of staining
 Explain the theory of staining
 Classify stains used in staining of histological material
 Describe the various histological dyes
 Prepare and use the stains
 Describe types of staining methods
 Describe mounting of stained sections.

Introduction
This is the process of imparting colour to tissue section in order to make evident

various tissue components. Staining can be achieved by using one stain, sequences of
stains or combination of stains to render one or more elements evident in contrasting
colours.
Stains are adopted to stain the general tissue structures like the nucleus and cytoplasm
or made specifically to stain tissue elements that are either intracellular or
extracellular.
Dye:
A dye is a substance that may be natural or synthetic used to impart colour to a
material. Dyes usually obtained from plants or animals are referred to as natural dyes
while those manufactured artificially are referred to as synthetic dyes. Dyes in
solutions are known as stains.
Classification of stains:
Chemically stains are salts. They can be basic, acid or neutral depending on whether
the colouring part or component is contained in the base or acidic part of the stain.
Acid stain:
The colouring substance is contained in the acidic radical of the dye and the basic part
is colourless. Acidic stains include Eosin, Acid fuchsin and orange G
Basic stain:
The colouring substance is contained in the basic radical while the acid part is
colourless, basic stains include Methylene blue and Haematoxylin and Basic fuchsin.

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Neutral stains:
Both the acidic and basic components are coloured. They are usually formed from the
precipitate that proceed a reaction between aqueous acid and basic stains when they are
mixed together ,examples are the Romanowsky stains such as Leishman, Giemsa and
May Grunwald..
Natural dyes:
These are dyes obtained from plants and animals specifically insects. They include: -
Haematoxylin, Carmine, Orcein, Litmus, Saffron and Brazilin.

Haematoxylin:

Chemical structure of haematoxylin

This is a natural dye obtained from a Mexican tree known as haematoxylon

compechianum. The dye is extracted by use of ether.
Haematoxylin has poor staining properties and is usually used in conjunction with a
mordant.
The staining element in haematoxylin is haematin which is obtained by oxidizing the
dye. This process of oxidation is referred to as ripening and is done by:-
 Exposure of the stain to air and sunlight for period of time depending on the
type of haematoxylin.
 An oxidizing agent like mercuric oxide and sodium iodate can be added to
speed up the oxidation process.
Haematoxylin dye is used to stain general tissue structures. Haematoxylin stains are
prepared in various ways depending on the mordant used. Mostly used mordants are
Aluminium and iron salts.

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All staining methods where the dye is taken up by the tissue structures without
addition of a mordant is referred to as direct staining. When a mordant is applied is
called indirect staining.
Mordant:
It is a metallic substance which facilitates a staining reaction by forming a link
between the dye and the tissue. The mordant combines with the dye to form a lake
which in turn binds onto the tissue.
Mordants are usually applied in three ways:-
 Mordant can be used during staining where it is applied before the stain;
this is referred to as pre-Mordanting an example is Heidenhain’s iron
haematoxylin.
 It can also be used in conjunction with the stain where it is called
metachrome as seen in Erlich’s alum haematoxylin
 The mordant may also be applied after the where it is referred to as post-
Mordanting for example in gram’ staining.
Histological mordants include:
- Iron (ferric ammonium sulphate, ferric chloride).
- Aluminium (ammonium aluminium, potassium aluminium sulphate and sodium
aluminium sulphate)
- Tungstate ( Phosphotungstic acid)
- Chromium
- Iodine
Alum Haematoxylin:
The mostly used alums are
- Ammonium aluminium sulphate
- Potassium aluminium sulphate.
- Sodium aluminium sulphate
- The mordant is incorporated into the stain during its preparation.
- They either contain a ripened or are exposed to air and sunlight to ripen for
several days.
Examples of alum haematoxylin are:-
- Erlich’s, Harris, Mayer’s, Coles and Delafield
Iron haematoxylin:
It is uses iron salts as mordants. The mostly used mordents are: - Ferric ammonium
sulphate, Ferric chloride.
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Mordents are not incorporated into the stains during preparation but are either used
before mixed with the stain just before use or used after the stain.
Examples of iron haematoxylins are:-
- Heidenhain’s, Weighert’s, Loyez, Verhoeff’s and Celestine blue

Basic differences between Alum and Iron haematoxylins

Alum Haematoxylin Iron haematoxylin
1. Mordant and stains are mixed together 1. Mordant and stains are kept separately to
during preparation. avoid over oxidation by the mordant.

2. Resultant colour is blue – purple 2. Resultant colour is black

3. Short staining time ( 5 -20 min) 3. Longer staining time
4. Differentiation of tissue not very critical 4. Differentiation of tissues critical
5. Less tissue structures are demonstrated 5. Several tissue structures are demonstrated
because of degree in differentiation and staining
time.
6. Colour fade from tissue section after a 6. Colour on section stay for many years
short time

i) Orcein.
- It is a dye extracted from lichens by action of ammonia and air.
- It is useful in histology during demonstration of hepatitis antigens and elastic fibres
tissue
ii) Litmus.
- It is obtained from lichens by action of ammonia air and lime (Potash or soda).
- It is a poor dye. Its use as a dye has long been abandoned but is currently used as an
indication
iii) Saffron:
- It is extracted from the stigmas of a plant referred to as crocus sativa.
-It is used as a counter stain but not commonly used in histology as it has poor staining
properties on tissues.

iv) Carmine (Cochineal)

-Extracted from bodies of female cochineal bugs.

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- The staining element is carmine which is obtained after treatment of the dye with
alum
- It is useful in the demonstration of glycogen, Neuropathology and zoological
Synthetic dyes.
- They are benzene derivatives. These dyes are organic compounds consisting of
benzene ring to which is attached both chromophoric and auxochromic group.
- Chromophores: Is the part of the dye that gives it colour.
-Auxochrome: - Is that part of the dye that gives it salt forming capacity
- Synthetic dyes include:
- Picric acid, Aniline dyes, such as crystal violet, methyl red and Methylene blue.

Picric acid structure

-NO2 (Nitro group) is a Chromophore

- OH (Hydroxyl group) is an Auxochrome.
- If an Auxochrome is lacking the substance is referred to as Chromogen which is able
to impart colour that can easily be removed. A Chromogen is not a dye an example is
trinitrobenzene that has a Chromophore but lacking an auxochrome.
Dyes are classified according to the chromophoric structure as follows;
a) Nitroso group
b) Nitro group
c) Azo group
d) Quinoid group
e) Thiazol group
f) Phenol methane group
g) Xanthene group
Types of staining method
There are several staining methods they include:
Vital staining
- This involves the staining of living cells. The cells can either be stained inside or
outside the body. This type of staining can be divided into two:-
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 ii. Intra – vital staining
iii. Supra –viral staining.
Intra- viral staining:
- It involves the staining of living cells while still inside the body.
- Vital dyes are injected into the body before the tissue is removed for microscopic
examination.
- The cells will ingest the dye such that when removed and examined they are seen to
be stained.
- This method is good during demonstration of Reticulo-endothelial cells
- Intra viral dyes include: - Indian ink, Congo and Tryphan blue
Supra – vital staining:
- This involves removal of a tissue form the body and stained immediately when cells
are still living.
- This type of staining is good during demonstration of mitochondria and
chromosomes during cell divisions supra vital dyes include –
i. Janus green.
ii. Methylene blue
iii. Neutral red
Elective solubility:
- This involves staining of structures by the dye moving from the original solvent to
the structure. Some dyes are known as Lysochrome dyes are more soluble in tissue
structures than the original solvent.
- Nearly all Lysochrome dyes are dissolved in alcohol.
- These dyes are mostly used in the demonstration of lipids (fats) Lysochrome dyes are
more soluble in fat than alcohol.
Lysochrome dyes include:-
ii. Sudan III
iii. Sudan IV
iv. Sudan black
v. Oil red O

Histochemical staining:
- This involves a chemical reaction between ion radicals in the stain and the tissue
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- It uses colourless or pale solutions called Leuco dyes to produce coloured substances
in the tissue.
- The ion radicals in the solution will react with the ion radicals in the tissue to give a
coloured substance usually a precipitate.

Examples
a) Periodic acid Schiff’s reagent which is colourless when added onto tissue is
oxidized to produce a coloured substance. It is used in demonstration of
glycogen.
b) Perl’s Prussian blue technique whereby potassium ferricyanide which is
pale yellow reacts with hemosiderin (iron) in tissue to form a blue
precipitate.
c) Feulgen reaction used for demonstration of DNA which produces a purple
colour
Metallic impregnation:
This involves the deposition of salts of heavy metals onto tissue elements; the salt is on
or around the tissue element. This differs from normal staining in that all elements
being demonstrated appear larger than normal. It cannot be classified as true staining
because it has the following characteristics
a) Structures so demonstrated are usually rendered opaque and black
b) The colouring matter is particulate
c) The deposit is on or around but not in the element so demonstrated
Certain tissue constituents have some property of reducing some metallic compounds
to the metallic state, producing an opaque usually black deposit
Ammonical silver solutions [Ag (NH 3)2] are commonly used for impregnation
technique.
They differ from stains in being colourless; they are simply deposited on the surface of
the tissue element or bacteria which in turn appears larger and more visible.
Tyrosine derivatives such as melanin and phenolics compounds such as Kultschitzky
cells of the intestinal glands have the capacity of reducing ammonical silver to form a
visible deposit.

Argentaffin substances:

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These are substances that have the capacity to reduce silver salts in the dark without
the aid of a reducing agent. Examples are Argentaffin cells in the intestinal mucosa,
melanin, uric acid and basement membrane (derived from a Latin word Argentum
which means silver and therefore Argentaffin means ‘having affinity for silver’)
Argyrophilic substances:
‘Argyros’ in Greek means silver and argyrophilic means ‘silver loving’. Substances
that are impregnated by silver with the aid of a reducing agent or light are referred to
as argyrophilic substances they include; Reticulin fibres, calcium and spirochaetes.
Silver nitrate impregnation techniques:
 Gordon and sweet’s – Reticulin fibres
 Gomori’s silver technique Reticulin fibres
 Foot’s method Reticulin fibres
 Mason Fontana – Melanin and Argentaffin cells
 Von Kossa- Calcium demonstration
 Warthin – Starry method - Spirochaetes
 Grocott’s methenamine and Gridley’s silver methods - Fungi
General precautions (Silver techniques):
1. Sections tend to float off the slides due to the high alkalinity of the silver solutions
used and adhesives should be applied to slides.
2. Glassware used must be chemically clean because dirt acts as a reducing agent.
3. Light acts as a reducing agent and so all silver solutions must be kept in brown
bottles away from light.
4. Distilled water must be used because tap water contains impurities that may reduce
silver nitrate.
Theory of staining:
There are two theories that govern staining. They are the physical and chemical
theories.
Physical theory
The staining of tissue elements by dyes is governed by
a) Permeability
b) Solubility
c) Adsorption
d) Density

Density and Permeability:

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This involves the cell arrangement of the tissue. The more the cells the more fibrous
the tissue and the more it takes in the dye than the less fibrous tissue. Once the dye
penetrate the tissue dense tissue retain the stain more than less dense tissues.
Solubility
- Some dyes are more soluble in the tissue than the original solvent e.g. some dyes are
more soluble in fat than in alcohol e.g. Lysochrome dye.
Adsorption
Small substances get attracted to larger substances and get attached onto their surfaces
Chemical theory:
- When a dye is in solution it ionizes forming the acid and basic radicals. The acid
radical of the stain will stain the basic part of the tissue while the basic radical stains
the acid tissue structure.
Application of stains:
- Stains can be applied on tissue either progressively or regressively
Progressive staining:-
- It is selective staining of tissue where no differentiation is required. The tissue
structures are stained in a definite order, so that at the end of the staining period a
differential colour is attained. Time is very important.
Regressive staining:-
- It involves over staining of tissues with a stain then differentiated.
- The tissue structures are all stained the same so they must be differentiated for
individual structures to be visible. The structures that have affinity for the initial stain
will retain the colour of the dye while other structures become colourless and will be
stained by a counterstain.
- There are substances which when incorporated with the stains increase the staining
power and selectively of the solution. These substances are referred to as accelerators,
an example is phenol in carbol fuchsin and sodium hydroxide in Methylene blue.
- When used in demonstration of central nervous system they are referred to as
accentuates like ammonium bromide in cajal’s method for demonstration of axis
cylinder in nervous system
Factors responsible for staining variations.
2. pH of the solvent of the stain
3. Temperature at which the staining is administered
4. Nature of tissue e.g. density, binding sites

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5. Age of the stain, some stain best when fresh while others provide good staining
when they stay a little longer.
6. Solubility of the stain. Some stains have the ability to dissolve in certain tissue
structures than the original solvent.
7. Time, certain tissue structures take up stains within the shortest time possible and
others require longer period to stain.
8. Concentration of the stain
Guidelines on preparation and storage of staining reagents (Solutions).
Purchase
It should be done according to use such that reagents used more often should be
stocked in large amounts neither while those nor used routinely are stocked in small
quantities.
Dates
Dates of preparation and expiry date must be shown clearly staining tie and any
necessary precaution must be noted.
Storage:
- All dyes must be stored in a cool dry place or as directed by manufacturer
- Enzymes preparation (Solutions) must be kept in the refrigerator
- Silver solutions must be kept in dark places
- Most dyes and solutions are kept at room temp
- Osmic acid, aldehyde fuchsin are stored at 4oC
Storage containers
a) Store reagent in hard glass which may be brown or clear
b) Store reagent in plastic bottles where applicable to avoid breakage.

Control
Test new batches of dyes using known positive and negative materials before using
them
Toxicity
a) Assume that all dyes are toxic and so care must be followed when handling them
b) Avoid skin contact by use of gloves, forceps protective clothing etc.
c) Exercise great care to avoid spillage.
Accuracy
a) Use accurate balances when weighting.
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b) Avoid rough estimates
Solvents
Use specified solvents to dissolve the dyes or chemicals
Filter reagents after preparation and before use especially the stain
Labels
- Label all your reagents and give clear instructions on the use and disposal.
Demonstration of general tissue structures:
Haematoxylin and Eosin Method
Reagents:
 Tap water
 Xylene for dewaxing
 Alcohol (various grades)
 1% acid alcohol differentiators
 Eosin stains the cytoplasm red to pink
 Alum haematoxylin stain – stain nuclei blue
 Mountant DPX preserves tissue and raise R. index.
 Scott’s water optional) for bluing – tissue become available.
Ingredients of Scott’s water
Sodium bicarbonate – 7 g
Magnesium sulphate – 40 g
Tap water – 200 ml
1% formalin (few drops) preservative
Other requirements:
1. Staining rack
2. Staining jars
3. Cover slip
4. Gauze/cotton wool
5. Tissue section on slide.
Fixation: Most tissues that require demonstration of general tissue structures are fixed
in 10% formal saline.
Principle:
- Haematoxylin stain is a basic stain and it stains the nuclear because it is acidic while
eosin being an acid stain is taken up by the basic tissue structure like the cytoplasm.
- The dye and the mordant form a lake which then attaches on to the tissue structures.
Method:
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 1. Dip tissue section in 2 – 3 changes of xylene for about 7 minutes (2 -3 minutes
in each ) this is to remove the paraffin wax from the tissue to allow stain
penetration
2. Transfer the tissue to 2 changes of absolute alcohol followed by descending
grades such as 90%, 70% and 50%. Treatment with absolute alcohol removes
the xylene from the section because it does not mix with water.
Descending grades of alcohol are used to avoid shrinkage of tissue section if
taken to water directly. This is to orientate the tissue to water which is a solvent
of most stains.
3. Wash in tap water to remove alcohol
Note: Steps 1 -3 are usually referred to as bringing sections to water or dewaxing
and hydrating.
4. Flood the section with haematoxylin stain or dip in staining jar containing the
stain for 5 -20 minutes. This is to stain the acidic structures in the tissue to
allow visual recognition or clarity.
5. Rise in tap water to remove excess stain
6. Differentiate in 1% acid/ alcohol for up to 30 seconds to remove excess stain.
7. Blue in running water for 10 minutes or Scott’s tap H 2O) for 30 seconds. This
is to make the tissue structures become alkaline for easy staining with Eosin
and also to make nucleus to be bluer.
8. Counterstain in Eosin for 3 -5 minutes to give colour contrast to haematoxylin
stained structures to allow comparison of structures. This is to stain structures
not stained by the initial stain and thus providing contrast.
9. Dehydrate in ascending grades of alcohol such as 30%, 70%, 90% and
absolute. This is to remove the water which will not mix with xylene.
10. Clear in 2 -3 change of xylene if it is to be mounted in a hydrocarbon
containing mountant.
11. Mount in DPX- This is to preserve and protect the tissue section (from dust and
pests) and also to raise the refractive indeces of various tissues structures for
ease identification.
Expected results:
Nuclei – blue – Purple
Cytoplasm – Red to pink
Red blood cells- Orange
Rest of tissue – shades of red pink
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Stained Tissues sections

(Liver tissue) (Intestine)

(Skeletal Muscle)

(Cardiac Muscle)

(Smooth Muscle)

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Alum haematoxylins in common use.
Mayer’s haematoxylin.
Has a life span of 2 – 3 months
Ingredients
 Haematoxylin (Dye )– I gram
 Ammonium aluminium sulphate salt (mordant) – 50 grams.
 Water (solvent) – 1,000 millilitres
 Sodium iodate (ripening agent )– 0 -2 grams
 Acetic acid (nuclear sharpener) – 20 millilitres
 Chloral hydrate (preservative) -50 grams
Harris haematoxylin:
 Life span – months – years
 Haematoxylin (dye) – 5 g.
 Ammonium alum (Mordant) – 100g
 Ethanol ( solvent) – 50 ml
 Water ( solvent )– 1,000ml
 Mercuric oxide (ripener)- 2.5 g
 Acetic acid – sharpener – 40 ml
Delafield’s:
1. Life span – several years
2. Haematoxylin ( dye )– 6 g
3. Ammonium alum – 60 g
4. Ethanol – 50 ml(solvent
5. Glycerol – 150 ml (Preservative)
6. Water – 600 ml (solvent

Erlich’s:
 Life span – several years
 Haematoxylin – 6 g
 Ammonium – 40 g
 Ethanol – 300 ml
 Glycerol – 300 ml
 Water – 300 ml

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  Ripening agent – 0.64g (sodium iodate /mercuric oxide) potassium
permanganate. can also be used as a ripener
 Acetic acid – 32 ml

Coles
 Life span – several years
 Haematoxylin – 1.5 g
 Water – 250 ml
 Ammonium alum – 70 g
 Ripening agent (any oxidising agent may be used).

Mounting of sections:
- This is a process of covering the section with a suitable medium and cover slip,
when the medium dries it makes a permanent preparation which can be examined
without damaging the section.
Mounting media are syrupy fluids that are used between the coverslip and the
tissue. They are used to protect the tissue from dust, pests and to increase the
refractive index.
- The choice of the mounting media depends on the staining procedure used. All
the mountants have refractive indices close to that of glass, mounting media can be
divided into two main groups
o Aqueous mountants
o Resinous mountants.
a) Aqueous mountants:
These are mountants which contain water as a solvent. They are meant to mount
tissue sections that have been stained with water containing reagents/solutions.
Mounting can either be temporary or permanent. The formula of these mountants
include the following
1. A solidifying agent such as. Gelatine and gum Arabic
2. An agent to prevent cracking on drying glycerol.
3. An agent to raise the refractive index like cane sugar
4. A preservative like phenol, arsenic trioxide, thymol and sodium methiolate.
5. Solvent used is water.
There are various aqueous mountants in use. The common ones are
 Glycerine jelly

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  Apathy’s
 Hyman’s
 Farrant’s
 Karo corn syrup
 Laevulose ( Fructose )syrup
Glycerine Jelly:
 Has a refractive index of 1.47
Composition:
Gelatine – 10 g solidifying agent
Glycerine – 70 ml anti cracking agent
Dist H2O – 60 ml solvent
Phenol – 0.25 g – preservative
Preparation:
- warm the gelatine at 60oC till it dissolves in distilled H2O
- Add glycerol and phenol and mix well
- Label and store at 4oC
- For use melt at 60oC
- Good for frozen sections
ii) Apathy is Mountant:
 Refractive index is 1.52
 Used to mount sections for fluorescence microscopy
Composition:
 Gum Arabic – 50 g (solidifying agent)
 Cane sugar – 50 g (raise refractive index)
 Dist. H2O – 50 ml (Solvent)
 Thymol – 0.05 g ( preservative)
iii) Hyman’s (modification of apathy’s)
 Refractive index is 1.52
 Used to mount sections stained with Metachromatic stains
Composition:
 Gum Arabic – 50g (solidifying agent)
 Can sugar –20g (Raise refractive index)
 Potassium acetate – 10 ml (buffer)
 Sodium methiolate – 0.5 g (preservative)
 Dist. H2O– 40 ml (solvent)
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iv) Farrant’s Mountant
 Refractive index is 1.43
Composition:
 Gum Arabic – 50 ml ( solidifying agent)
 Glycerol – 50 ml (anti -cracking agent)
 Arsenic trioxide – I g (preservative), Sodium methiolate can also be
used as a preservative
v) Karo corn syrup
 Refractive index is 1.47
Composition
 Karo con syrup – I volume
 Distilled H20 2 volumes
 Thymol – I crystal
vi) Laevulose (Fructose) syrup
 Refractive index is 1.47
 On long standing it forms crystals so it must be used immediately
Composition
 Fructose - 70 gm
 Distilled H2O– 20 ml.
b) Resinous Mountants;
-These are mountants that contain a resin dissolved in a hydrocarbon solvent such as
xylene
- The resin can either be natural or synthetic.
- They are used to mount sections which have been dehydrated and cleared in a
hydrocarbon solution
b) Natural Resinous mountants:
The resins that are used obtained from trees. The most commonly used are
a) Canada balsam
b) Colophonium resin
c) Damar balsam
i) Canada balsam
- Has a refractive index of 1.52
- It is obtained from a Canadian fir known as abies balsamea
- Can be prepared to have an acid or neutral reaction
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Preparation (Neutral).
- Dissolve 50 -60% balsam in xylene
- Add calcium carbonate in excess and stir thoroughly.
- Filter.
- Then let xylene evaporate in the incubator/ evaporation of xylene is
controlled
- The colour is brown.
Acid reaction
- Prepared like the neutral reaction
- Salicylic acid is added instead of calcium carbonate
ii) Colophonium resin
 Has a refractive index of 1.52
 Solvent used is xylene or turpentine.
 When dissolved in alcohol it can be used as a differentiator in Giemsa staining
during demonstration of Leishmania donovani bodies
iii) Damar Balsam
 Refractive index 1.53
 Obtained from an East Indian tree known as Sherea weisneri
Preparation
- Dissolve the balsam in chloroform then evaporate the chloroform
- Then dissolve in xylene
c) Synthetic resinous mountants
- These are mountants prepared from synthetic (Artificial) resins. They are
available commercially
They include:-Euparol Mountant, DPX. (Dibutyl phthalate xylene), Xam and BPS.
Euparol Mountant has a refractive index of 1.48
- It is a semi-synthetic commercial product
- Is available in two shades of colour (colourless and greenish/ bluish.)
- The greenish/bluish shade is due to addition of copper salt.
- Good for unstained sections to provide a clear background
Composition: Sandarac resin, Eucalyptus, Paraldehyde and Camsol salt.
DPX (refractive index 1.52)
Composition
- Polystyrene resin (Disterene 80), Dibutylphthalate and Xylene.
Xam (R.I- 1.52)
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 - Has two colours
- Pale yellow (this allows mounting of unstained tissues as the yellow
colour gives contrast).
- Colourless
Characteristics of a good Mountant
1. Should harden quickly.
2. Should not cause aniline (basic) dyes to fade
3. Should not crack or develop granules on hardening
4. Should be clear with a refractive index as close to that of glass as possible.
5. Should be free from dirt, moulds, dust etc.
6. Should not contain air bubbles.
Ringing media:
- These are used to surround when the mountant is fluid to prevent it from evaporation.
- It is used mostly in temporary preparations that have used aqueous mountants.
-It immobilizes the cover slip and prevents attachment of slides on storage.
Examples of ringing media include:
a) Paraffin wax
b) Dunoyers wax.
c) Kroning cement
d) Asphaltum nail varnish.
Kroning cement – 10 parts paraffin wax and
40 parts Colophonium resin

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 Chapter Ten:

Histological Pigments:
Learning outcomes
At the end of the instruction the learner should be able to:
 Define Histological pigment
 State the importance of the pigments
 Describe the classification of the pigments
 Discuss the various histological pigments
 Outline the various demonstration methods

Introduction:
These are coloured substances found in tissues both in normal and pathological
conditions. They occur as a result of processing in certain solutions such as fixatives,
formed in the body during metabolic activity or enter the body from the outside
environment. The importance of having knowledge on the pigments is related to the
following:
 Presence of pigments in tissues may mask the structures to be studied hence
there is need to know their characteristics. This will help in determining
precautions to be taken to prevent them and how they can be removed when
encountered.
 Some of the pigments when present in the tissues indicate a pathological
condition thus helping in the diagnosis of disease.
Classification of pigments:
Pigments can be divided into; (i) Artefact or fixation, (ii) exogenous and (iii)
endogenous pigments.
(i) Artefact pigment
These are groups of pigments that are introduced into tissues during tissue processing.
Common pigments are introduced during fixation in certain fixatives. They include the
following; Formalin, Mercuric chloride, Osmium tetroxide, Potassium dichromate,
Pink’s artefact disease and stain deposits (precipitate).
Formalin pigment

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It is also referred to as post mortem pigment. The pigment is seen in tissues that were
fixed in acid formaldehyde containing fixatives such as formalin and formal sublimate.
This pigment is mostly observed in blood containing tissues like Liver, spleen and
bone marrow which have stayed for at least 24 hours before fixation after removal
from the body. The breakdown of red blood cells releases haematin a derivative of
haemoglobin which combines with acid formaldehyde to form the pigment.
Formation of the pigment can be avoided by immediate fixation of the tissue when
removed from the body and by use of Neutral buffered formal saline or non-
formaldehyde containing solutions.
Characteristics of the pigment
The pigment is brown/black in colour and birefringent under polarised light. It is
soluble in alcoholic picric acid and alcoholic alkaline solutions. Removal of this
pigment from the tissue is based on the following methods; Schridde’s, Varocay’s and
Barrett’s.
Schridde’s method
-Bring section to 70% alcohol
-Treat with alcoholic ammonia solution for 30mins
-Wash thoroughly in tap water
-Continue with the staining procedure.
Varocay’s method
-Bring section to 80% alcohol
-Treat tissue with alcoholic potassium or sodium hydroxide for 10 minutes
-Wash in water
-Treat with 80% alcohol for 5 minutes
-Wash in water and proceed with staining technique required.
Barrett’s method
-Deparaffinize section in xylene
-Wash thoroughly in absolute alcohol
-Treat with saturated alcoholic picric acid for 30 minutes
-Wash in absolute alcohol to remove picric acid
-Bring section to water and continue with staining technique.
Mercuric chloride pigment
This is another pigment that occurs in tissues that have been fixed in mercuric chloride
containing fixatives like Formal sublimate, Helly’s fluid and Zenker’s fluid.

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It is dark brown in colour and soluble in lugol’s iodine. The pigment is therefore
removed from tissues by treating it with the iodine.
Method of removal
-Bring section to water
-Immerse tissue in lugol’s iodine for 5 minutes
-Bleach in 5% sodium thiosulphate until it is colourless (5 minutes)
-Rinse thoroughly in water and proceed with staining technique required
Note; Lugol’s iodine can also be added in the processing alcohols and the pigment will
be removed when the section is being brought to water.
Osmium tetroxide pigment
The pigment is seen in tissues that have been fixed in osmium tetroxide containing
fixatives such as Fleming’s fluid. It is black in colour and can be removed from tissues
by bleaching in hydrogen peroxide –alcohol mixture or potassium permanganate
solution.
Method of removal
Bring section to 70% alcohol
Bleach section in a mixture of hydrogen peroxide and 70% alcohol
Expose it to sunlight for 30 minutes
Rinse thoroughly in water and continue with the relevant staining technique
Or
Bring section to water
Transfer to 0.5% potassium permanganate
Rinse in water
Rinse in sulphurous rinse to remove the permanganate
Wash thoroughly in water and continue with the staining procedure
Dichromate /chromic acid pigments
They are imparted into tissues by any fixative containing dichromate like Zenker’s,
Helly’s, Orth’s and Muller’s fluids. The pigment is yellow brown in colour and is
removed by overnight washing in water or by use of 1% acid alcohol followed by
washing in water.
Stain precipitate
This imparted into tissues during staining due to faulty technique or over oxidation of
staining solutions. Improper washing and leaving the stain to dry on the slide is a
contributory factor. It can be prevented by proper washing of the tissue during

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staining. Staining solutions must be filtered after preparation or just before use to
remove the deposits or crystals that may form.
Pink’s artefact disease
This is the inability of haematoxylin to stain the cell nucleus. The nucleus takes the
eosin thus appearing pink.
The occurrence is mostly seen in tissues that are rapidly fixed in 10%buffered formal
saline. It is commonly observed in lymphoid tissues and epithelial cells.
It can be avoided by addition of 1% acetic acid to the fixative or 1% acid alcohol to
dehydrating alcohols.
(ii) Exogenous Pigments:
These are pigments that gain access accidentally and serve no physiological function.
Mostly they are of mineral origin. They enter the body through inhalation,
implantation and rarely ingestion.
The pigments are mostly found in the lungs, skin, lymphoid tissues and visceral
organs. These pigments include: Carbon, Silica, and Asbestos.
Carbon:
The pigment is seen in the lungs, lymphoid tissues skin and some visceral organs as
black particles.
It is a non-reactive pigment and therefore cannot be demonstrated by any known
histological method.
It cannot dissolve in acids, alkalis, alcohol or any other solvent.
Silica:
Found in tissues removed from people who are involved in mining. The sites that are
affected include; the Lungs and Lymphoid tissues. It causes fibrosis of the lungs in a
condition known as Silicosis.
It has the following characteristics;
Resistant to micro-incineration
Birefringent under polarised light
Can be detected by use of autoradiography
Asbestos:
This is a purified form of silica that is found deposited in the same sites as silica and
carbon.
Found as long-beaded rods in tissues. It causes asbestosis a condition characterised by
fibrosis.

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The pigment is birefringent and when coated with protein and hemosiderin it looses
birefringence. It is also resistant to micro-incineration. It can be demonstrated by Perl’s
Prussian blue method as it gives a positive reaction.
(iii) Endogenous pigments
These are coloured substances produced within the tissues and which serve a
physiological function or are bye-products of body metabolic processes. They are
divided into;
a) Haematogenous pigments
b) Autogenous pigments

Endogenous
pigments

Non-
Haematogeno haematogenou
us s
(Hemoglobin)

Haem Globin Lipidic Non-lipidic

Malaria
Iron pigment
Protein part Lipofuchsin Ceroid
Bile pigments Melanin

Deposited in
Used in new Bilverdin
tissues as Amino acid
synthesis
hemosiderin Bilirubin pool

Haematogenous pigments

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These are pigments that present in tissues as a result of blood related physiological
processes. They include; Hemazoin (malaria pigment), Hemosiderin (free iron),
Hematoidin (Bile pigment) and Haemoglobin.
(ii) Malaria pigment
This pigment is also referred to as Hemazoin pigment and found in tissues after an
infection with malaria parasites. The pigment appears dark brown in colour and is
intracellular mostly seen in liver, spleen, brain Phagocytic and red blood cells.
Removal of the pigment from tissues can be done by use alcoholic picric acid
(Barrett’s method).
(ii) Hemosiderin (free iron)
This is a constituent of haemoglobin which is golden yellow. In tissues it is found
bound with protein and can exist as ferric or ferrous salt, the most abundant is the
ferric salt.
It is soluble in acids but not soluble in water, alcohol or any alkaline solution. Under
normal conditions small amounts are found in the liver and spleen. Large amounts are
found in tissues in conditions such as Haematochromatosis, Chronic venous
congestion and indiscriminate absorption of iron from the small intestine.
Hemosiderin can be demonstrated by the following methods:
 Perl’s Prussian blue for ferric salts
 Turnbull’s for ferrous salts
Perl’s Prussian blue
Requirements:
 Xylene
 Various grades of alcohol
 Distilled Gauze
 Tissue section on a slide
 4% hydrochloric acid
 4% Potassium ferrocyanide
 1% neutral red
 DPX Mountant
 Staining racks
 Staining jars

Principle

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Ferric ions (Iron III) Combines with potassium ferrocyanide to form an insoluble
precipitate ferric ferrocyanide which is blue in colour. The ferric protein bound is
broken by 4% hydrochloric acid to allow the reaction to take place.
Method
1. Bring section to distilled water
2. Apply a mixture of equal volumes of 4% hydrochloric acid and 4% potassium
ferrocyanide for 15minutes
3. Rinse in distilled water
4. Counterstain in 1% Neutral red for 30 seconds-3 minutes
5. Dehydrate in ascending grades of alcohol
6. Clear in 2 changes of xylene and mount
Results:
Ferric salts – Blue
Other tissue structures – Red
Precautions:
 Tap water should not be used because it may contain some traces of iron that
will give false positive results. Use distilled or deionised water.
 Preparation of reagents should also be done using distilled or deionised water.
 All containers must be chemically clean
 Avoid metal containers and metal racks

Turnbull’s Method
Requirements
The requirements are those of Perl’s Prussian blue except the following;
20% potassium ferricyanide used instead of 4% potassium ferrocyanide and
2% Hydrochloric acid instead of 4% Hydrochloric acid
Principle;
This method involves formation of a blue precipitate by the interaction of ferrous salts
with 20% potassium ferricyanide to form ferrous ferrocyanide.
3FeCl2 +2K3Fe (CN) 6 – Fe3 [Fe (CN) 6]2 +6KCl
Method:
- Bring section to water and rinse in distilled water
- Treat in freshly prepared solution of equal parts of 20% potassium ferricyanide and
2% hydrochloric acid
- Rinse in distilled water
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- Counterstain in 1% neutral red
- Dehydrate in ascending grades of alcohol
- Clear in xylene and mount in DPX
Expected results:
Ferrous salts – Blue
Other structures – Red (colour of the counterstain)
Autogenous pigments:
These are substances produced in the body and are not as a result of destruction of
blood. They include: Melanin, Lipofuchsin, Argentaffin, calcium, copper and
chromaffin cells.
Melanin:
This is a brown-black pigment produced from tyrosine. An enzyme tyrosinase acts on
tyrosine to produce a substance called dihydroxyphenilalanine (DOPA), the enzyme is
also referred to as DOPA oxidase. The same enzyme acts on DOPA rapidly to produce
an intermediate pigment which polymerises to produce melanin.
This pigment is bound to proteins which are located in the cytoplasm of tissue cells
commonly found in the skin, eye and the brain.
Melanocytes (cells containing melanin) produced by the skin are found scattered in the
basal layer of epidermis
Melanophages are found in the upper dermis of the skin in inflammatory diseases of
the skin.
In the eye the pigment is found in the choroid, ciliary body; iris and retina.
The pigment is found in variable quantities in pathological conditions such as benign
and malignant tumours (melanoma).
Melanin can be demonstrated in tissues by the following methods
(a) silver reducing methods like Masson Fontana
(b) Solubility and bleaching
(c) DOPA oxidase detection
(d) Florescent microscopy
(e) Schmorl’s method

Masson Fontana
Requirements:
1. Xylene
2. Various grades of alcohol
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 3. Grams iodine
4. 5% Sodium Thiosulphate
5. Ammonical silver nitrate
6. 2% gold chloride
7. 0.1% Saffranin
8. Distilled water
Principle:
This is a metallic impregnation method that is dependent upon its ability of certain
phenolics compounds or tyrosine derivatives to reduce silver solution to its metallic
silver.
Method:
1. Bring section to water
2. Treat with grams iodine for 10 minutes
3. Transfer to 5% sodium thiosulphate for 2 minutes
4. Wash in distilled water
5. Leave overnight in ammonical silver nitrate solution in the dark in a
closed jar
6. Tone briefly in 2% gold chloride for 2 minutes
7. Rinse in distilled water
8. Fix in 2% sodium thiosulphate for 2 minutes
9. Wash in running tap water for 2 minutes
10. Counterstain in 0.1% Saffranin or 1% neutral red for 2 minutes
11. Wash in water then 70% alcohol
12. Dehydrate in alcohol clear in xylene and mount in DPX.
Results:
Melanin – Black (Intracellular)
Argentaffin granules - Black
Nuclei and other structures – Red
Lipofuchsin:
This is a pigment that is also referred to as brown atrophy or wear and tear. It is
yellow brown in colour and mostly found in the liver and cardiac muscles cells ( when
found in muscle cells is known as brown atrophy).
The pigment is as a result of oxidation and breakdown of lipid and lipoprotein in aged
persons but when seen in tissues of younger persons are considered pathological.
There are various methods of demonstration but the most reliable is Schmorl’s method.
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Schmorl’s method:

Principle:
The procedure is dependent on reduction of ferricyanide to ferrocyanide with the
production of ferric ferrocyanide which is blue in colour.
Procedure:
1. Bring section to distilled water
2. Treat with a mixture of 1% potassium ferricyanide and 1% ferric
chloride
3. wash in 1% acetic acid
4. Wash in water
5. Counterstain in 1% neutral red for 3 minutes
6. Dehydrate rapidly in alcohol
7. Clear in xylene and mount in a neutral mountant

Results:
Lipofuchsin – Dark blue (Extra-cellular)
Argentaffin granules – blue
Melanin – Dark blue Intracellular
Other structures – red
Argentaffin cells:
They are also referred to as enterochromaffin cell granule or Kultschitzky cells. The
cells are found throughout normal alimentary canal and are best seen in the pyloric
glands of the stomach and mucosa of the appendix. Argentaffin cells have affinity for
silver salts and chrome salts.
Argentaffin cells contain very little 5-hydroxytryptamine but are produced in large
amounts in carcinoid tumours of the gut and lungs. For proper demonstration, a
dichromate fixative in conjunction with formalin is used. Formalin converts 5HT into
tetrohydro-carboline derivative which is responsible for the staining reactions.
Demonstration of the cells is by any of the following methods: Masson Fontana,
Schmorl’s and Diazo techniques.
Diazo technique:
To obtain correct results the tissue must be fixed rapidly informal saline and later
treated with 3% potassium dichromate.
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Solutions:
a) Fast red salt B- it contains 5mls of 15 aqueous fast red B salt and 2mls of saturated
aqueous carbonate. During preparation it is placed in a refrigerator at 4-5 oC for 10
minutes.
b) Harris haematoxylin
Principle:
Fresh stabilized fast red B solution demonstrates enterochromaffin cells by colouring
the green – red while haematoxylin acts as a nuclear counterstain.
Procedure:
1. Bring section to distilled water
2. stain in solution (a) in the refrigerator for 2 minutes
3. Rinse in distilled water
4. Stain in solution (b)
5. wash in running tap water for exactly 3 minutes
6. Blue in tap water for 10 minutes
7. Dehydrate in ascending grades of alcohol
8. clear in xylene
9. Mount in DPX

Results:
Argentaffin cell granules – orange to red
Nucleus – Blue
Positive Controls:
Tissues that are removed from small intestine, carcinoid tumours or appendix all fixed
in formalin.
Chromaffin cells:
These cells contain granules that have affinity for chrome salts. They become brown
when fixed in dichromate solutions.
The cells are normally found in adrenal gland and related organs. Large amounts of the
cells are seen in pneachromocytoma tumour of the adrenal gland. The cells contain
high amounts of adrenalin and noradrenalin hormones and their precursors.
Fixation:
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Tissues must be fixed promptly in dichromate fixative such as Clampy’s fluid,
Muller’s fluid and Regaud’s fixative overnight. They must be freshly prepared and pH
adjusted to 5-6.
(Regauds fluid contains 3% aqueous potassium dichromate and 40% formaldehyde
solution).
Staining methods:
1. Giemsa stain
2. Schmorl’s reaction
3. Periodic acid Schiff’s (PAS)
Principle:
Regaud’s fixative change to greenish in colour on standing, it therefore imparts a green
colour on the chromaffin tissue. Giemsa stain is neutral and will stain the nuclei blue
with the red blood cells taking a bright pink colour.
Technique:
1. Bring section to water
2. Stain in Giemsa diluted 1:10 overnight
3. Differentiate in 1% acetic acid to remove excess pink stain
4. Treat with 1% potassium hydroxide in 95% alcohol to remove excess blue
colouration and to intensify the pink stain
5. Control stage 3 and 4 microscopically
6. When the right colours are achieved dehydrate rapidly in 3 changes of alcohol
7. Clear in xylene and mount in DPX.
Results:
Chromaffin – Green blue
Nuclei – Blue
Red blood cells – Bright pink

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 Chapter Eleven:
Bacterial demonstration in tissues:

Learning Outcomes:
At the end of the instruction the learner should be able to:
 Fix tissues for demonstration of bacteria
 Differentiate the various bacteria that infect human tissues
 Prepare reagents and solutions used for demonstration of bacteria
 Outline staining methods for bacterial demonstration
 Microscopically examine and report on stained tissues

1. Gram stain:
The method differentiates bacteria in gram positive and negative organisms due to
their differences in the cell wall. Some bacteria after the uptake of the basic stain resist
decolourization by acetone
Tissues requiring demonstration of bacteria by gram staining method in tissues are
normally fixed in 10% formal saline though any other fixative can be used. Paraffin
wax tissue sections of 3-5 microns are ideal.

Staining Solutions and Reagents.

a) Crystal violet:
Ingredients
Crystal violet – 2gm
95% alcohol - 20mls
Ammonium oxalate – 0.5gm
Distilled water – 30mls
Preparation: Dissolve crystal violet in 95% alcohol. Dissolve ammonium oxalate in
distilled
Aniline dyes like methyl and Gentian violet can also be used instead of crystal violet.

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b) Grams iodine
Iodine – 1gm
Potassium iodide – 2gm
Distilled water – 200mls

c) Aniline/xylene mixture
Aniline – 2 parts
Xylene - 1part
d) 0.5-1% Neutral red or saffron

Principle:
Crystal violet stains gram positive bacteria blue while the counterstain neutral red
stains gram negative bacteria red.
Method
1. Dewax the tissue section in xylene/aniline mixture
2. Pass the section through descending grades of alcohol
3. Wash the section in water
4. Stain in crystal violet for 2-3 minutes
5. Wash off stain with grams iodine then leave on the slide for 2- 3 minutes
6. Decolorize in acetone
7. Rinse in water
8. Counterstain in neutral red or in any other counterstain for 2- 3 minutes
9. Rinse in water
10. Blot dry and rapidly dehydrate in aniline/xylene mixture
11. Clear in xylene and mount in DPX
Results:
Gram positive bacteria – Blue
Gram negative bacteria – Red
Tissue structures – Red

2. Acid Fast Bacilli

Bacilli causing tuberculosis and Leprosy belong to this group. Their cell wall contains
a light lipid known as mycolic acid. The cell wall confers acid fastness or the ability to
resist discoloration.

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Mycobacterium tuberculosis or tubercle bacilli cause tuberculosis while
mycobacterium leprae causes leprosy.
Mycobacterium tuberculosis is both acid and alcohol fast while mycobacterium leprae
is only acid fast.
Tissues that require demonstration of mycobacterium may be fixed in most of the
fixatives except those containing fat solvents like Carnoy’s fluid to avoid dissolving of
the mycolic acid. Due to inability of mycobacterium leprae to resist decolourization by
alcohol all fixatives with alcohol must be avoided.
Mycobacterium tuberculosis:
This can be demonstrated in tissues by the following methods; Ziehl Nelsen and
fluorescent microscopy.
Ziehl Nelsen Method:
Application of 0.5% ammonium hydroxide before of formalin fixed tissue sections
improves the brightness of the colour of the acid fast bacilli. Due to the alkalinity of
the solution care must be taken because it may detach the section from the slide.
Staining Solutions and reagents
a) Carbol fuchsin:
Basic fuchsin – 1gm
Absolute alcohol – 10ml
5% phenol – 100ml
b) 3% acid alcohol or 20% sulphuric acid
c) Counterstain: Methylene blue (mostly used) or Malachite green

Methylene blue – Methylene blue – 1gm

- Glacial acetic acid – 1ml
- Absolute ethyl alcohol – 20ml
- Distilled water – 80ml
Principle:
Mycolic acid which surrounds the bacilli enables the uptake of carbol fuchsin and
resists acid and alcohol decolourization. Phenol acts as an accelerator and facilitates
the penetration of the dye.

Method:
1. Remove paraffin wax by use of xylene
2. Using descending grades of alcohol remove xylene
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 3. Wash in water
4. Place a rectangular paper over the tissue section on a staining rack or filter the
stain before use and flood the section for 30 minutes
5. Rinse in tap water
6. Decolorize in 25% H2sO4 or 3 % acid alcohol until the section is pale pink or
no more stain comes. This takes about 5minutes
7. Counterstain in malachite green or methylene blue for 2 -3 minutes
8. Rinse in water
9. Blot dry or briefly dehydrate in alcohol to avoid the counterstain getting
washed off.
10. Clear in xylene and mount in DPX
Expected results
Acid alcohol fast bacilli (AAFB) – Brick red
Other tissue structures- Blue or green depending on the counterstain used
Red blood cells – Pale pink
Mycobacterium leprae:
The mycobacterium is acid fast but not alcohol fast like mycobacterium tuberculosis
because it has less mycolic acid in its cell wall. Due to its inability to resist alcohol
decolourization the tissue should not come in contact with alcohol.
During staining of the organism weak concentration of sulphuric acid is used.
The demonstration method of choice is Wade fite method or simply referred to as Fite
method.
Staining solutions and reagents
Staining solutions remain the same as Ziehl Nelsen method except the decolourizer
alcohol and the dewaxing agents
a) Turpentine and kerosene mixture
b) Carbol fuchsin
c) 10% sulphuric acid
d) Methylene blue
Principle:
Mycolic acid which surrounds the bacilli enables the uptake of carbol fuchsin and
resists acid and alcohol decolourization. Phenol acts as an accelerator and facilitates
the penetration of the dye.
Procedure:
1. Dewax tissue section with 2 changes of turpentine and alanine mixture
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 2. Blot to remove excess deparaffinizer
3. Wash in water for 5 minutes
4. Filter Carbol fuchsin onto the section or put on a rectangular filter paper on the
section and flood with the stain for 15-30 minutes
5. Rinse with tap water and decolorize with 10% H2SO4 for 3 minutes
6. Rinse with water and counterstain with methylene blue or malachite green for
1-2 minutes
7. Drip dry or blot with filter paper and place in an incubator at 56oC
8. Clear in xylene and mount in DPX
Expected results:
Mycobacterium leprae (AFB) – Bright red
Other tissue structures – Blue or green depending on the Counterstain
Red blood cells – Pale red

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 Chapter Twelve:
Demonstration of Connective tissue
Learning outcomes
At the end of the instruction the learner should be able to:-
 Describe the characteristics of connective tissue fibres
 State the principles of all demonstration methods
 Outline the various staining methods
 Prepare and use staining solutions
 Examine microscopically and interpret the results

Introduction:
Connective tissue fibres that are normally demonstrated to provide diagnosis of certain
diseases that affect them, they are; Collagen, elastic and Reticulin fibres. These fibres
are products of connective tissue cells called fibroblasts.
In scurvy disease there is reduction in collagen fibres while there is an increase in
rheumatoid arthritis.
Elastic fibres are demonstrated in case such as calcification of tissues as in old age,
cases of asthma, diseases affecting liver, spleen and lymphoid tissue.
a) Collagen fibres:
They are also referred to as white fibrous tissues because they appear colourless. The
fibres are found where great strength is required like in tendons and ligaments. They
are long fibres that are usually in bundles without branching and are flexible but not
elastic.
The fibres contain Gelatin that is often seen when boiled and when hydrolysed they
yield glycine.
Collagen fibres are dense, tough and as such needs long and careful processing to
ensure thorough impregnation. The fibres are digested or are swollen when treated
with weak acids and alkaline solutions.
They can be demonstrated by: (a) Celestine blue, Haematoxylin and van gieson
sequence (b) Weighert’s Van gieson and (c) Masson trichrome staining methods.
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Weighert’s Van Gieson Method
Staining Solutions

1. Weighert’s haematoxylin
Solution A
Haematoxylin dye – 1gram
Absolute alcohol–100ml. (Ethanol)
Solution B
30% Ferric chloride - 4ml.
Distilled water - 995ml.
Concentrated hydrochloric acid – 1 ml.
(For use mix equal parts of solutions A and B)
2. Van Gieson
1% aqueous acid fuchsin - 10ml.
Saturated aqueous picric acid- 90ml
Mix and stand for three weeks.
Procedure
Principle
Acid fuchsin in Van gieson stains collagen fibres red by hydrogen bonding while
picric acid differentiates the haematoxylin stained structures and colours muscle
yellow. Haematoxylin stains the nucleus grey to black. Ferric chloride acts as a
mordant.
Method:
 Bring section to water
 Rinse in absolute alcohol
 Stain in Weighert’s haematoxylin for 15 minutes
 Wash in running tap water
 Counterstain in Van gieson for 3minutes
 Rinse in 95% alcohol
 Dehydrate in 3 changes of absolute alcohol
 Clear in 3 changes of xylene
 Mount in DPX mountant

Expected results:
Nucleus – grey to black
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Collagen fibres- red
Smooth muscle – yellow
Red blood cells – yellow

c) Elastic fibres:
These fibres appear yellow in fresh tissue and therefore are referred to as yellow elastic
fibres. They occur singly or as branched to form a network. Elastic fibres are highly
elastic and are found where strength and ability to stretch is required like in the walls
of the blood vessels, lungs, ligaments vocal cords and skin.
They consist of elastic fibres that yield glycine and leucine when hydrolysed, resist
digestion by pepsin enzyme but, can slowly be digested by trypsin enzyme.
The fibres are resistant to autolytic changes and can be calcified in disease or in old
age.
Microscopic examination is possible when demonstrated by any of the following
methods.
 Verhoeff’s elastic method
 Aldehyde fuchsin
 Orcein
 Weighert’s elastic fibres
Mostly used methods are Verhoeff’s and Aldehyde methods
Verhoeff’s elastic method
Solutions
1. Verhoeff’s iodine (mordant) –Potassium iodide (4g), Iodine (2g) and 100ml of water
Preparation: Dissolve potassium iodide in 5ml water, dissolve iodine in this solution
and make the volume to 100ml.
2. Solution A: 5% Alcoholic haematoxylin- stain
3. Solution B: 10% Ferric chloride- mordant
4. 2% Ferric chloride- differentiator
5. Van gieson – Counterstain
Verhoeff’s haematoxylin
5% Alcoholic haematoxylin-20ml
10% Ferric chloride-8ml
Verhoeff’s iodine – 8ml
The mixture is done just before use.
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Principle:
Iodine and ferric chloride acting as mordants form a “lake” on the nuclei and elastic
fibres to which haematoxylin attaches thus staining them.
Method
 Bring tissue section to water
 Stain in Verhoeff’s haematoxylin for 15 minutes
 Rinse in water
 Differentiate in 2% ferric chloride for 10 minutes (control microscopically to
avoid over differentiation)
 Transfer to 95% alcohol to remove the colour of iodine
 Wash in water and Counterstain in van gieson
 Dehydrate in quick rinses of alcohol
 Clear in xylene and mount in DPX
Expected results:
Elastic fibres – grey to black
Cell nucleus – grey to black
Collagen fibres – red
Muscle fibres – yellow
Red blood cells – yellow
Aldehyde Fuchsin method
Solutions
Basic fuchsin – 0.5g
70% Alcohol - 100ml
Concentrated hydrochloric acid – 1ml
Paraldehyde – 1ml
Preparation:
-Dissolve the dye in alcohol and add the acid and paraldehyde
-Leave at room temperature to ripen for 24-72 hours
-Store in the refrigerator at 4oC
-Prepare fresh every 3 months
Principle:
A combination of basic fuchsin and paraldehyde in presence of a strong mineral
acid (HCl) reacts with sulphated mucopolysaccharides such as elastic fibres, mucus
and mast cells colouring them purple
Method
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  Bring section to water
 Wash in 70% Alcohol
 Stain in aldehyde fuchsin for 10 minutes
 Differentiate in 95% alcohol
 Counterstain in van gieson for 3 minutes
 Rinse in absolute alcohol for 3 minutes
 Dehydrate in alcohol, clear in xylene and mount in DPX.
Expected results:
Elastic fibres – purple/violet
Collagen fibres – red
Reticulin fibres
These fibres are formed in the lymphoid tissue and other solid organs like liver and
spleen. They are fine branching fibres that form a framework to support cells in
these organs.
They have very little elasticity and are resistant to weak acids and alkalis. The
fibres consist of Reticulin fibres that produce glycine when hydrolysed and are
never digested by pepsin or trypsin enzymes.
They are demonstrated by special silver methods under metallic impregnation
techniques.

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Summary of properties of connective tissue fibres:
Collagen Elastic Reticulin
Appearance Colourless in fresh Yellow in fresh Only demonstrated
tissue hence known tissues hence known by special silver
as white fibres as yellow fibres methods known as
argyrophilic fibres
Distribution Widely distributed Blood vessels, Lungs, Lymphatic system,
particularly in skin, vocal cords and liver and spleen
tendons, joints and ligaments
ligaments
Refractive index Low High Low
Pepsin digestion Rapid Resistant resistant
Trypsin digestion None Slow None
Structure Long fibres in wavy Fine fibres which Short fibres which
bundles no branching branch to form a branch to form a
network close network
Chemical Collagen yields Elastin yields glycine Reticulin yields
composition glycine on hydrolysis and leucine on glycine on hydrolysis
and yields collagen hydrolysis
on boiling
Haematoxylin, Pink - -
eosin staining
Silver Brown with some - Black
impregnation techniques
Van gieson red Yellow -
Masson trichrome Green - Green
Orcein - Dark brown -
Verhoeff’s Red black None
Aldehyde fuchsin Red Purple -

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 Chapter Thirteen:
Cytology:
Learning outcomes
At the end of the instruction the leaner should be able to:
 Define cytology
 State the application of cytology in human health
 Describe the stages of cells
 Name the various cytological specimens
 Discuss cytological fixatives
 Explain the various staining methods
 Outline the characteristics of malignant cells
 Explain the classification of pap smears
 Describe postage of cytological specimens to referral laboratories
 Describe characteristics of cancer cells
 State types of tumours

Introduction
Cytology is the microscopic study or examination and interpretation of cells in order to
confirm their normal and abnormal characteristics. The study of cells shaded from
body surface is known as exfoliative cytology.
The shedding or exfoliation can be spontaneous from body epithelial surface or may be
removed from such surfaces or membranes by physical means. Such cells are usually
shedded into effusions or secretions from the lining epithelial layer.
This shedding occur due to normal epithelial surface which because of constant
growth continue to shed old cells from the superficial layers as they are replaced by
new cells. Cells can also be shedded as a result of pathological conditions that make it
necessary for the affected cells to be removed from the body.

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During cell life exfoliation occur due to the cell undergoing different stages. Those
stages are:
1. Birth stage - Occur at the basal or reserve cells where cells multiply through
the process of mitosis.
2. Differential or maturation stage – Cells differentiate into various body cells
3. Functional stage - Cells are now mature and are able to perform various body
functions
4. Degenerative stage- Cell function decreases followed by cell death. This is
where the cell is shedded off.
Normal epithelial cell layer is composed of the following cells
a) Superficial
b) Intermediate
c) Parabasal
d) Basal.

Superficial

Intermediate

Parabasal

Basal
Basement Membrane

Normal cells depending on the site and function have some structures that are uniform
such as the nucleus, mitochondria, and endoplasmic reticulum; cells from the same site
usually have the same shape and size.
- The role of cytology has the following application:-
o Detection of malignancy
o Assessment of hormonal function/infertility
o Identification of infection and inflammatory cells.
o Used in medical legal cases such as investigation of rape.
a) Malignancy.

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 This is the abnormal growth of cells. These abnormal cells can be detected in
specimen collected from the cervix, fallopian tubes, ovaries, vaginal wall,
vulva, mouth (Buccal cavity) and thorax. Secretions such as sputum, urine,
gastric juice, C.S.F, pleural fluid, ascetic fluid, breast secretions and fine needle
aspirations (FNA) can contain malignant cells. Microscopic examination of
these specimens may be used as a screening method for detection of early
cancer.
b) Hormonal function:
The cytological assessment of the endocrine functions depends on the fact that
changes in the hormonal balance are reflected in the vaginal and cervical
epithelia. The most common amplification is in the investigation of infertility,
threatened abortion and non-pregnancy amenorrhea. Microscopic examination
of vaginal or cervical cells may reveal the various stages of cell maturation and
can assist in assessment of oestrogen and progesterone hormones.
Male infertility can also be assessed through tests such as semen analysis and
testosterone assessment.
c) Infections:
Protozoan infection by Trichomonas vaginalis and the fungus Candida albicans
are commonly diagnosed through examination of specimens from the female
genitalia. Bacterial, Viral and Fungal infections may be determined through
examination of smears made from deposits of secretions from certain body
organs such secretions include, urine, pleural fluid, C.S.F. and sputum.
Presence of shedded cells from the organs may be an indication of micro-
organism infection, cells like Neutrophils and Histiocytes are some of those
seen in micro-organism infections.
d) Medici- legal:
Through examination of cells certain disputes or cases may be solved.
Examination of spermatozoa recovered from the vagina of a rape victim is an
important cytological use and DNA studies are very crucial in in solving
paternity disputes.
Medico-legal can be defined as a science that uses a case of injury, ailment, etc,
by investigation agencies to evaluate information such as cytological and other
information to fix responsibility regarding the cause of injury or ailment.
Advantages of Cytology:

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 1. It is cheap and simple (no much reagents is required to carry out diagnostic
investigation).
2. The processing methods of specimens are not long hence immediate results
obtained
3. There is no serious injury to body tissue because no surgical procedures on the
patient.

Cytological specimens
These are samples that are collected from the patient/client for analysis. If any
cytological specimen is to be useful it must be representative (typical) of the body site
sampled whether normal or abnormal. Detecting abnormal cells is the outcome of a
series of interdependent samplings of specimens to produce credible results. This can
be achieved by making sure that every step is done very carefully from the collection
of specimen to production of results. The following are key steps to be undertaken:
1. The specimen collection technique that samples the biologic
process.
2. The cyto-preparatory samples the specimen already collected.
3. The screening process samples the preparation, involves proper
identification of the sample and proper techniques.
4. The morphological interpretation samples the cellular features.
The pap test was introduced into clinical practice in the mid 1940’s and is now
heralded as the most successful cancer screening test in medical history. The are two
types of pap tests; conventional, yesterday’s pap test and liquid-based preparations,
today’s pap test. The conventional pap test was the standard of practice for decades.
Conventional Pap test:
In the beginning only plain glass slides without a frosted label end were available.
According to papanicolaou the materials needed were: Clean slides, labelled
beforehand with a piece of index card 1inch square fastened to one end of the slide by
a paper clip. On the label the name of the patient, the date and the type of smear were
written.
The original pap tests were either vaginal aspirates or swab smears that were taken
either a non-absorbent swab or a wooden Ayre spatula.
Conventional pap tests presented with certain limitations. Majority of false-negative
conventional pap tests were due to clinical sampling errors, screening errors and
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interpretation errors. Counts of epithelial cells on conventional smears showed that
only a fraction of the available epithelial cells on the sampling devices were actually
deposited on the slides.
Liquid-Based preparation:
The term-liquid-based preparation’ was introduced in 1998. It involves rinsing the
sampling implements in liquid preservative, which is then transported to the
laboratory. The specimen is homogenized and a subsample thin-layer preparation is
made. There are two liquid-based preparations; ThinPrep Pap test (TPPT) and
SurePath Pap test (SPPT). They have replaced the conventional pap test as the standard
practice.
Rinsing the device that has been used to sample the cells in a liquid preservative is a
sure way of transferring all cells (100%) to allow sampling of the cells onto the slides.
This also makes sure that the sample is available for further slide preparation unlike
the conventional method where the specimen is used and discarded (Cellular material
gets lost when the devices like spatula is discarded).
Some brushes used have detachable broom head and this can be detached into the
preservative after sample collection thus making sure no cell is lost.

They are divided into

1. Gynaecological
2. Non- gynaecological
i) Gynaecological
These are collected from female genitalia.
There are several methods of collecting these specimens. Most collected
specimens include
Cervical layer scrape smear using an ayre spatula.
This involves collection of cells from cervix using a spatula or an endocervical brush a
broom-like sampling device
A smear is then made on a glass slide and stained for microscopy. The smear is
useful in the diagnosis of the cancer of the cervix.
Aspirate from posterior fornix
This involves collection of material from posterior fornix pool using a plastic suction
pump; a smear is made on a glass slide from the cells collected. It is useful in
diagnosis of the endometrial and the upper vaginal carcinoma.
Vaginal smears-
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The smears are taken using moist cotton wool on the tip of an applicator stick. This is
introduced into endocervical (vaginal) canal and rotated. The collected material is
then smeared onto a clean slide and fixed immediately. Vaginal cancers and detection
of infection micro-organisms are detected through examination of the smears.

Endometrial aspiration:
Under strict conditions a syringe is passed onto the uterine cavity, the material is then
aspirated with the syringe and is smeared onto a clean glass slide and fixed
immediately for staining and microscopy. This method offers high detection of
endometrial carcinoma.
ii) Non – Gynaecological
- These are specimens other than those of female genitalia. They include urine,
sputum, ascetic fluid, gastric juice, C.S.F, pleural fluid and buccal cavity smears.
Urine
- Examination of urine plays an important role in the detection of cancer of the urinary
tract, prostate and renal parenchyma. It can also indicate any infection of the urinary
tract by micro-organisms.
- For males any early morning urine specimen is satisfactory while for female it is
obtained by catheterization and obtaining midstream to obtain the best result.
- Urine is centrifuged and a smear is made from the deposit. It is then fixed
immediately.
Urine with low cellular content may require cytocentrifugation.
Sputum:
- Sputum is collected in a wide mouthed contained avoid saliva for best outcome of
examination.
- Smear is made and fixed immediately then later stained.
- It is important in detection of lung and respiratory tract cancer.
Gastric, pleural and ascetic fluids.
- They are aspirated from different parts of the body. They are then processed as urine.
- They contain protein and some coagulate on being removed, to avoid coagulation
they can be collected in a container with 3.8% sodium citrate.
- The specimens are useful in the diagnosis of carcinomas, hodgins disease and
tuberculosis.
Impression smear
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-These are made by cutting the tumour and touching it with a clean slide. It is then
fixed immediately.
Cerebrospinal fluid (CSF).
- Its examination is useful in the diagnosis of tumour in the central nervous system and
infections by organisms such as Cryptococcus, Neisseria and pseudomonas bacteria.
- It has very little cells and these cells can be harvested by sedimentation onto a clean
slide by use of a special centrifuge known as cytocentrifuge.
- The cells are then fixed immediately.
Cytological fixatives
Fixatives used for cytological smears are:-
a) Equal volume of 95% ethyl alcohol and ether mixture
b) 95% ethyl alcohol
c) Mixture of 7 parts tertiary butyl alcohol and 3 parts 95% ethyl alcohol
d) Schaudin’s fluid composition
– absolute ethyl alcohol
– Glacial acetic acid
– Saturated aqueous mercuric
chloride
- All cytological material must be fixed immediately (when still wet) they are
collected. This is because cells degenerate very fast and cellular details are distorted.
- The fixative used must be capable of penetrating rapidly with good preservation of
cell morphology.
- All cytological fixatives are applied on smears for at least 15 minutes.
-Low temperatures can also be used to preserve the specimen but for a short time only.
- Specimens which have no protein material in them can easily wash off the slide
during staining. This can be avoided by applying an adhesive onto the slide before
smear preparation
Postage of smears:
- Smears for postage should be covered using a synthetic resin consisting of diaphane
resin and 95% ethyl alcohol.
- The slide is flooded with the resin which is then left to dry leaving a hard smooth
film over the smear. The film is removed on arrival to the laboratory by applying
alcohol either mixture for 20 -30 minutes.
- They can also be protected by use of carbowax 1500 which is a mixture of carbowax
1500, absolute alcohol and glacial acetic acid. When it dries it forms a hard protection
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over the smear. The wax can be removed by treatment with ethyl alcohol Aerosol
sprays (fixatives) are also available but are expensive. The force of the spray may
displace the cells off the slide.
They are usually polythene glycols in alcohol. If they are not available normal
fixatives can be used dried then packed nicely.
Staining methods for cytological smears.
1. Methylene blue (good for screening purposes especially for sputum).
2. Periodic acid Schiff’s for body fluids.
3. Perl’s Prussian blue –for sputum.
4. Giemsa stain (body fluid and sputum)
5. Shorr’s method – ( hormonal assessment )
6. Feulgen reaction– ( DNA)
7. Papanicolaou method – good for all cytological smears. It is most popular
because it gives excellent nuclear details and makes the cytoplasm
translucent.
Methylene blue method:
It is a wet film technique for malignant screening purposes. It is useful in busy
laboratories especially for sputum.
Solution: - 0.5% Methylene blue aqueous.
Principle
Methylene blue being a basic dye stains the acid nucleus.
Method:
1. Place 2 -3 drops of a centrifuged deposit on a slide.
2. Add equal volume of 0.5% Methylene blue
3. Using a hot plate or flame warm the slide slightly while mixing the
specimen and the stain.
4. Continue warming until the fluid and the stain evaporates ( do not allow the
slide to dry).
5. Cover with a cover slip and examine (the stain fades after I hour).
6. Keeps long if the stain is mixed with glycerine or if the edges of the cover
slip are sealed with paraffin wax or any ringing media.
Results:
Nucleus – shades of blue
Cytoplasm –pale blue
Nucleus of malignant cell – Dark blue.
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 Papanicolaou staining method.
Solutions:
1. Harris haematoxylin
2. Orange G which consists of – Phosphotungstic acid and 0.5% orange in
95% alcohol.
3. Eosin Azure which consist of:-
a. 0.5% light green SF in 95% alcohol
b. 0.5% Bismarck brown 95% alcohol
c. 0.5% Eosin Y in 95% alcohol
d. Phosphotungstic acid
e. Saturated aqueous Lithium carbonate.
Principle:
Haematoxylin stains the nucleus by dye mordant tissue complex formation
Eosin azure being an acid stain will stain the cytoplasm whereby eosin-Y has affinity
for mature cells while light green SF has affinity for immature (young) cells.
Orange G also being an acid stain has an affinity for the cytoplasm of the old
superficial cells.
Method:
1. Make smears and fix when still wet in appropriate fixative
2. Hydrate smears by passing through descending grades of alcohol.
3. Rinse in distilled water.
4. Stain in Harris haematoxylin for 4 minutes
5. Rinse in H2O
6. Differentiate in 1% acid alcohol (1-5 sec).
7. Blue in running tap H2O for 10 minutes or Scott’s tap water for 30 seconds.
8. Rinse in 95% alcohol
9. Stain in orange G for 2 minutes
10. Rinse in 95% alcohol
11. Stain in eosin azure for 5 minutes
12. Rinse in 95% alcohol
13. Dehydrate in absolute alcohol
14. Clear in xylene
15. Mount in DPX
Results
Cytoplasm – Mature cells – orange to red
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 Intermediate cells –bluish to green
Young cells (Basal and Parabasal) – green to blue
Nucleus (all cells) - blue
Red cells - variable colour
Fungi - Orange – red
Trichomonas vaginalis - bluish
Nucleus of malignant cells – deep blue

Papanicolaou stained cells

Microscopic examination:
Examination of stained smear is important in diagnosis of infections and malignancy.
For diagnosis of malignancy the following are taken into consideration
a) Background (site) of cells.
b) Arrangement of cells
c) Number cells
d) Shape and size of cells.
Each individual cell is then examined as follows.
1. Nucleus – Shape and Size
- Nuclear /cytoplasm ration
- Chromatin distribution
- Position.
- Number and size of nucleoli
- Staining characteristics
2. Cytoplasm
- Nuclear /cytoplasm ration
- Vacuoles
- Shape
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 - Presence and absence
3. Staining characteristics

Cancer Cells
Cancer cells like normal cells have the nucleus and cytoplasm and fulfil all
conditions for cell production and propagation. They differ with normal cells in
that they have the following characteristics.
i) They have marked tendency to separate from one another due to low
calcium content.
ii) They vary in shape and size due to crowding and rapid growth due to mass
production.
iii) The features of the nucleus and cytoplasm are as follows
a) Cytoplasm
It does not resist physical pressure and therefore the cell form odd
cytoplasmic shapes and at times it disintegrates completely leaving the
nucleus alone. It may contain vacuoles.
b) Nucleus:
i) There is enlargement causing a decrease in nuclear /cytoplasm ratio
(there is no increase in the overall size of the cell).
ii) There is irregular nuclear outline with variation in shape and size.
iii) There is marked hyperchromasia due to increase in DNA which
stains deeply with basic dyes.
iv) The cell has multiple nuclei (several nuclei in one cell)
v) Uneven distribution of chromatin
Classification of cytological smears (pap smears).
Class I
All the cells in the smear shows no abnormality
Class II
Smear shows abnormality in the cells, the abnormality is not due to malignancy. It
may be due to inflammation or degeneration change like infection with bacteria,
Candida, Trichomas vaginalis and mechanical damage or allergic reactions
There is usually an increase in White blood cells, histiocytes and mast cells.
Class III
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Cells in this class have some signs of malignancy but are not definate; they are referred
to as suspicious or borderline cells.
Class IV:
The cells have distinct characteristics of malignancy
Classification of cervical smears. (Pap smear.)
Smear from the cervix can be classified as cervical neoplasm (CIN) classification is as
follows.
CIN O- Normal smear
CIN I- Mild dysplasia
CIN II - Moderate dysplasia (Same as class II)
CIN III- Severe dysplasia (Same as class II)
CIN IV- Carcinoma “insitu”
CIN V - Invasive squamous cells carcinoma (Spreading).
The Bethesda Classification system:
The Bethesda System (TBS) is a system for reporting cervical or vaginal cytologic
diagnoses, used for reporting Pap smear results. It was introduced in 1988, and revised
in 1991 and 2001. The name comes from the location (Bethesda, Maryland) of the
conference that established the system.

Types of results

Abnormal results include:

Atypical squamous cells
a) Atypical squamous cells of undetermined significance (ASC-US)
b) Atypical squamous cells - cannot exclude HSIL (ASC-H)
c) Low grade squamous intraepithelial lesion (LGSIL or LSIL)
d) High grade squamous intraepithelial lesion (HGSIL or HSIL)
Squamous cell carcinoma
a) Atypical Glandular Cells not otherwise specified (AGC-NOS)
b) Atypical Glandular Cells, suspicious for AIS or cancer (AGC-neoplastic)
c) Adenocarcinoma in situ (AIS)

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Squamous cell abnormalities

LSIL - Low-grade Squamous Intraepithelial Lesion

Low grade squamous intraepithelial lesion (LSIL or LGSIL) indicates possible cervical
dysplasia. LSIL usually indicates mild dysplasia (CIN 1), more than likely caused by a
human papillomavirus infection. It is usually diagnosed following a Pap smear.
CIN 1 is the most common and most benign form of cervical intraepithelial neoplasia
and usually resolves spontaneously within two years. Because of this, LSIL results can
be managed with a simple "watch and wait" philosophy. However, because there is a
12-16% chance of progression to more severe dysplasia, the physician may want to
follow the results more aggressively by performing a colposcopy with biopsy. In the
event that dysplasia progresses, treatment may be necessary. Treatment involves
removal of the affected tissue, which can be accomplished by LEEP, cryosurgery, cone
biopsy, or laser ablation.

HSIL - High-grade Squamous Intraepithelial Lesion

High grade squamous intraepithelial lesion (HSIL or HGSIL) indicates moderate or
severe cervical intraepithelial neoplasia or carcinoma in situ. It is usually diagnosed
following a Pap test. In some cases these lesions can lead to invasive cervical cancer, if
not followed appropriately.
HSIL does not mean that cancer is present. Of all women with HSIL results, 2% or less
have invasive cervical cancer at that time, however about 20% would progress to
having invasive cervical cancer without treatment. To combat this progression, HSIL
is usually followed by an immediate colposcopy with biopsy to sample or remove the
dysplastic tissue. This tissue is sent for pathology testing to assign a histologic
classification that is more definitive than a Pap smear result (which is a cytologic
finding). HSIL generally corresponds to the histological classification of CIN II or III.
HSIL treatment involves the removal or destruction of the affected cells, usually by
LEEP. Other methods include cryotherapy, cautery, or laser ablation, but none are
performed on pregnant women for fear of disrupting the pregnancy. Any of these
procedures is 85% likely to cure the problem.

Note:
 CIN- Cervical intraepithelial neoplasm
 Neo- New things formed

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  Dysplasia- Formation of abnormal tissue
 Neoplasm- New growth
 Metaplasia- Change of one kind of tissue to another
 Dyskaryotic- First stage of abnormality in cervical smear

Causes of cells abnormality

1. Genetic factors which are inherited from the family genealogy.
2. Chemical substances that include: Azo dyes, nitrosamines and hydrocarbon like
benzenes.
3. Infection with virus especially the DNA viruses that bring about mutation of the
body cells.
4. Physical agents such as exposure to radiations like X- rays infra-red and ultra
violet rays.
5. Hormonal imbalance excess hormone production may cause an abnormal cell
division.
6. Immunological surveillance (Immune suppression).
Treatment
a. Electrical burring of the affected part to destroy the abnormal cells
b. Cryotherapy – CO2 is passed to the affected area at high pressure to remove the
abnormal cells.
c. Surgery is performed when the abnormality has advanced to remove the affected
tissue
d. In very advanced cases especially the invasive types there is no treatment
False negative results.
1. Specimen collected form the working place.
2. Tumour cells falling to exfoliate
3. Unsuitable smear with the presence of blood cells
4. Technical error inexperienced person doing the examination
Management of follow up:
1. Smear unsuitable – repeat immediately
2. Normal findings – repeat yearly for routine check up.
3. Mild dyskaryosis – Repeat six months
4. Moderate dyskaryosis – Repeat after three months
5. Severe dyskaryosis -Biopsy taken for further confirmation.

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The cancer cell

The characteristics of normal cells

Normal body cells have a number of important characteristics. They can

 Reproduce themselves exactly

 Stop reproducing at the right time
 Stick together in the right place
 Self-destruct if they are damaged
 Become specialized or 'mature'

How cancer cells are different

Cancer cells are different to normal cells in several ways. They don't die if they move
to another part of the body and

 Cancer cells do not stop reproducing

 Cancer cells do not obey signals from other cells
 Cancer cells do not stick together
 Cancer cells do not specialize, but stay immature
 Cancer cells do not reproduce exactly the same as the original cell

Cancer cells do not stop reproducing

Unlike normal cells, cancer cells do not stop reproducing after they have doubled 50 or
60 times. This means that a cancer cell will go on and on and on doubling. So one cell
becomes 2, then 4, then 8, then 16....

The cancer cells may be able to stop themselves self-destructing. Or they may self-
destruct more slowly than they reproduce, so that their numbers continue to increase.
Eventually a tumour is formed that is made up of billions of copies of the original
cancerous cell. Scientists describe cancer cells as being 'immortal'.

Cancer cells do not obey signals from other cells

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Something in the cancer cells overrides the normal signalling system. This may be
because the genes that tell the cell to reproduce keep on and on sending signals. Or
because the genes that normally tell the cell to stop reproducing have been damaged or
lost. So the cancer cell keeps on doubling, regardless of the damage the extra cells
cause to the part of the body where the cancer is growing.

Cancer cells do not stick together

Cancer cells can lose the molecules on their surface that keep normal cells in the right
place. So they can become detached from their neighbours.

This partly explains how cancer cells spread to other parts of the body.

Cancer cells do not specialize, but stay immature

Unlike normal cells, cancer cells do not carry on maturing once they have been made.
In fact, the cells in a cancer can become even less mature over time. With all the
reproducing, it is not surprising that more of the genetic information in the cell can
become lost. So the cells become more and more primitive and tend to reproduce more
quickly and even more haphazardly.

What are Tumours?

The word cancer is derived from the Latin word for crab because it grabs onto
something and will not let go. The term cancer refers to a new growth which will
invade surrounding tissues, metastasize (spread to other organs) and may eventually
lead to the patient's death if untreated.

We often hear about cancer from friends and family and in the news. The terms tumor
and cancer are sometimes used synonymously which can be misleading. A tumor is not
necessarily a cancer. The word tumor simply refers to a mass. For example, a
collection of pus is by definition a tumor. A cancer is a particularly threatening type of
tumor. It is helpful to keep these distinctions clear when discussing a possible cancer
diagnosis.

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 An abnormal new growth of tissue that grows more rapidly than normal
cells and will continue to grow if not treated. These growths will compete
Neoplasm-
with normal cells for nutrients. This is a non-specific term that can refer to
benign or malignant growths. A synonym for tumor.
The more commonly used term for a neoplasm. The word tumor simply
Tumor- refers to a mass. This is a general term that can refer to benign or
malignant growths.
A non-malignant/non-cancerous tumor. A benign tumor is usually
Benign localized, rarely spreads to other parts of the body and responds well to
tumor- treatment. However, if left untreated, benign tumors can lead to serious
disease.
Malignant Cancer. A malignant tumor is resistant to treatment, may spread to other
tumor- parts of the body and often recurs after removal.
cancer- A malignant tumor (a malignant neoplasm).

Types of tumours
Benign tumours

Precancerous conditions

Malignant tumours

How tumours and cancers are named

Tumours are groups of abnormal cells that form lumps or growths. Different types of
tumours grow and behave differently, depending on whether they are non-cancerous
(benign) or cancerous (malignant). Precancerous conditions have the potential to
develop into cancer.

Benign tumours

Benign tumours are non-cancerous. They rarely cause serious problems or threaten life
unless they occur in a vital organ or grow very large and press on nearby tissues.

Benign tumours tend to grow slowly and stay in one place, not spreading into other
parts of the body.

Once removed by surgery, benign tumours don’t usually come back (recur). Benign
tumours usually stay non-cancerous, except in very rare cases.

Precancerous conditions

Precancerous (premalignant) cells are abnormal cells that may develop into cancer if
they are not treated. Some cells develop mild changes that may disappear without any
treatment. Other cells pass on genetic changes and new cells gradually become more
and more abnormal until they turn into cancer. It can take a long time for this to
happen.

Precancerous (or premalignant) changes can vary in their degree of abnormality.

 hyperplasia – an abnormal increase in the number of cells

o Some hyperplasias are precancerous, but most are not.
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  atypia (atypical) – cells look slightly abnormal under a microscope
o Sometimes atypia refers to changes caused by healing and
inflammation, rather than a precancerous change, and the cells go back
to normal once inflammation goes away or the body heals.
 metaplasia – cells look normal under a microscope, but are not the type
normally found in the that tissue or area
o Metaplasias are usually not precancerous.
 dysplasia – cells develop abnormally, have an abnormal appearance and are not
organized like normal cells
o Dysplasia almost always refers to a precancerous condition.

People with precancerous conditions are usually checked regularly, so they can be
treated quickly if cell changes become more severe.

Malignant tumours

Malignant tumours are cancerous. Cancer can start in any one of the millions of cells
in our bodies. Cancer cells have a larger nucleus that looks different from a normal
cell’s nucleus, and cancer cells behave, grow and function quite differently from
normal cells.

Malignant tumours vary in size and shape. They grow in an uncontrolled, abnormal
way and can grow into (invade) nearby tissues, blood vessels or lymphatic vessels.
They can interfere with body functions and become life-threatening.

Cancer cells can break off and spread to distant locations in the body (metastasize).
Cancer that spreads from its original location (the primary tumour) to a new part of the
body is called metastatic cancer. Malignant tumours can also come back (recur) after
they are removed.

How tumours and cancers are named

The most common way to name cancers is to do so according to the place in the body
they start, such as breast cancer or prostate cancer. Cancers of the blood are called
leukemia, while cancer of the plasma cells is called multiple myeloma and cancers of
the lymphatic system are called lymphoma. Some cancers have been named after the
person who first described them (for example, Hodgkin lymphoma or Wilms’ tumour).

Another way to name some benign or malignant tumours is after the type of cell or
tissue it develops from (tissue of origin). Most benign tumours and some malignant
tumours have the suffix "–oma" at the end of their name. When a malignant tumour
has the same name as a benign tumour, the word "carcinoma" or "sarcoma" is added to
the end to identify it as cancer. For example, a benign tumour of fatty tissue is called a
lipoma, whereas a malignant tumour of fatty tissue is called a liposarcoma.

How cancer spreads

Cancer cells can spread from where they started to other parts of the body, where they
can grow into new tumours. This process is called metastasis.

Cancer can spread in 3 ways:

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  Invasion (direct extension) – The tumour grows into surrounding tissues or
structures.
 Through the bloodstream (hematogenous spread) – Cancer cells break away
from the tumour, enter the bloodstream and travel to a new location in the
body.
 Through the lymphatic system – Cancer cells break away from the tumour and
travel through the lymph vessels and lymph nodes to other parts of the body.

Where cancer can spread

The type of cancer and where it starts often influences if and where it will spread. The
extent that cancer has spread when a person is diagnosed is called the stage. Many
cancers follow a staging system from 0 to 4 (IV). Knowing how and where a cancer
may spread helps doctors predict its possible course, plan treatment and further care.

These terms are also used to describe whether and how far cancer has spread:

 Localized – The cancer is confined to the original site.

 Regional spread – The cancer has grown into surrounding tissues or nearby
lymph nodes.
 Metastasis – The cancer has spread to a distant organ of the body or lymph
nodes far from the original (primary) tumour.

It is possible for cancer to spread anywhere in the body, but it is most likely to go from
its original site to other places in the body such as the bones, brain, liver or lungs.

Cancer Staging

Key Points

 Staging describes the extent or severity of a person’s cancer. Knowing the

stage of disease helps the doctor plan treatment and estimates the person’s
prognosis.
 Staging systems for cancer have evolved over time and continue to change as
scientists learn more about cancer.
 The TNM staging system is based on the size and/or extent (reach) of the
primary tumor (T), whether cancer cells have spread to nearby (regional)
lymph nodes (N), and whether metastasis (M), or the spread of the cancer to
other parts of the body, has occurred.
 Physical exams, imaging procedures, laboratory tests, pathology reports, and
surgical reports provide information to determine the stage of a cancer.

1. What is staging?

Staging describes the severity of a person’s cancer based on the size and/or
extent (reach) of the original (primary) tumor and whether or not cancer has
spread in the body. Staging is important for several reasons:

o Staging helps the doctor plan the appropriate treatment.

o Cancer stage can be used in estimating a person’s prognosis.
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 o Knowing the stage of cancer is important in identifying clinical trials
that may be a suitable treatment option for a patient.
o Staging helps health care providers and researchers exchange
information about patients; it also gives them a common terminology
for evaluating the results of clinical trials and comparing the results of
different trials.

Staging is based on knowledge of the way cancer progresses. Cancer cells grow
and divide without control or order, and they do not die when they should. As a
result, they often form a mass of tissue called a tumor. As a tumor grows, it can
invade nearby tissues and organs. Cancer cells can also break away from a
tumor and enter the bloodstream or the lymphatic system. By moving through
the bloodstream or lymphatic system, cancer cells can spread from the primary
site to lymph nodes or to other organs, where they may form new tumors. The
spread of cancer is called metastasis.

2. What are the common elements of staging systems?

Staging systems for cancer have evolved over time. They continue to change as
scientists learn more about cancer. Some staging systems cover many types of
cancer; others focus on a particular type. The common elements considered in
most staging systems are as follows:

o Site of the primary tumor and the cell type (e.g., adenocarcinoma,
squamous cell carcinoma)
o Tumor size and/or extent (reach)
o Regional lymph node involvement (the spread of cancer to nearby
lymph nodes)
o Number of tumours (the primary tumor and the presence of metastatic
tumours, or metastases)
o Tumor grade (how closely the cancer cells and tissue resemble normal
cells and tissue)
3. What is the TNM system?

The TNM system is one of the most widely used cancer staging systems. This
system has been accepted by the Union for International Cancer Control
(UICC) and the American Joint Committee on Cancer (AJCC). Most medical
facilities use the TNM system as their main method for cancer reporting.

The TNM system is based on the size and/or extent (reach) of the primary
tumor (T), the amount of spread to nearby lymph nodes (N), and the presence
of metastasis (M) or secondary tumours formed by the spread of cancer cells to
other parts of the body. A number is added to each letter to indicate the size
and/or extent of the primary tumor and the degree of cancer spread.

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 Chapter fourteen:
Semen Analysis:
Learning Outcomes
At the end of the instruction the learner should be able to:
 State the importance of semen analysis
 Describe collection methods of semen
 Describe the characteristics of normal spermatozoa
 State the normal accepted sperm parameters
 Identify abnormal spermatozoa

Introduction

A semen analysis evaluates certain characteristics of a male's semen and the sperm
contained in the semen. It may be done while investigating a couple's infertility or after
a vasectomy to verify that the procedure was successful. It is also used for testing
donors for sperm donation, in stud farming and farm animal breeding. Many men,
especially if they are older, have a semen quality analysis, testing for common
Sexually Transmitted Infections (STI), and perhaps testing for genetic defects done as
part of routine pre-pregnancy testing, though this is decisively not the norm, as most
doctors will not test the semen and sperm unless specifically requested or there is a
strong suspicion of a pathology in one of these areas discovered during the medical
history or during the physical examination.
Spermatozoa were first described by Leeuwenhoek in the 17th century, but, it was not
until 1928 that the sperm count was found to be associated with fertility potential.
Since that time a variety of sperm tests and semen parameters have been developed
with the hope of clarifying whether or not a man could impregnate his partner.
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 Semen analysis comprises a set of descriptive measurements of spermatozoa and
seminal fluid parameters that help to estimate semen quality.
Conventional semen analysis includes measurement of particular aspects of
spermatozoa such as concentration, motility and morphology and of seminal plasma.
Quantification and identification of non-spermatozoid cells and detection of anti-sperm
antibodies are also part of basic semen analysis.
Normal values of semen parameters issued by the World Health Organisation (WHO)
in 1992 are generally used as reference values.
Ideally, each laboratory should set its own normal values, reflecting the specific
population analyzed.
Normal values of semen variables (WHO 1992)
Standard tests
Volume 2.0 ml or more
pH 7.2-8.0
Sperm concentration 20x106 spermatozoa/ml or more
Total sperm count 40x106spermatozoa per ejaculate or more
Motility 50% or more with forward progression(categories a and
b)or 25% or more with rapid progression(category
a)within 60 minutes of ejaculation
Morphology 30% or more with normal forms
Vitality 75% or more live
White blood cells fewer than 1x106/ml
Immunobead test fewer than 20% spermatozoa with adherent particles
MAR test fewer than 10% spermatozoa with adherent particles
Optional tests
 -Glycosidase (neutral) 20 mU or more per ejaculate
zinc(total) 2.4  -mol or more per ejaculate
Citric acid(total) 52  -mol or more per ejaculate
Acid phosphatase (total) 200 U or more per ejaculate
Fructose(total) 13  -mol or more per ejaculate

Nomenclature for semen variables (WHO 1992)

Normozoospermia normal ejaculate as defined in table I
Oligozoospermia sperm concentration fewer than 20x106/ml
Asthenozoospermia fewer than 50% spermatozoa with forward
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 progression(categories a and b)or fewer than 25%
spermatozoa with category a movement
Teratozoospermia fewer than 30% spermatozoa with normal
morphology
Oligoasthenoteratozoospermia signifies disturbance of all three
variables(combination of only two prefixes can be
used)
Azoospermia no spermatozoa in the ejaculate
Aspermia no ejaculate

Normal semen is an admixture of spermatozoa suspended in secretions from the testis

and epididymis which are mixed at the time of ejaculation with secretions from the
prostate, seminal vesicles, and bulbourethral glands. The final composition is a viscous
fluid that comprises the ejaculate.

Sample collection and delivery

The most common way to collect a semen sample is through masturbation, directing
the sample into a clean cup. A sample may also be collected during intercourse in a
special type of condom known as a collection condom. Collection condoms are made
from silicone or polyurethane, as latex is somewhat harmful to sperm. Many men
prefer collection condoms to masturbation, and some religions prohibit masturbation
entirely. Adherents of religions that prohibit contraception may use collection
condoms with holes pricked in them.
A third option for collecting a sample is through coitus interruptus (withdrawal). With
this technique, the man removes his penis from his partner near the end of intercourse
and ejaculates into a cup.
Finally, if a blockage in the vas deferens is suspected to impede fertility, semen can be
taken directly from the epididymis. Such a collection is called per cutaneous
epididymal sperm aspiration (PESA).
The following instructions for sample collection and delivery are based on WHO
recommendations. The subject should be provided with clearly written or oral
instructions concerning the collection and, if required, transport of the semen sample.

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1. The sample should be collected after a minimum of 48 hours and no longer than 7
days of sexual abstinence. The name of the man, period of abstinence, date and time of
collection should be recorded. The time interval between the last ejaculation and
sample collection should be well defined and preferentially as constant as possible in
order to allow a reliable interpretation of the results of, in particular, sperm
concentration and motility. When the duration of abstinence is more than 7 days,
sperm motility, that is the proportion of spermatozoa with rapid progressive motility,
may decline. If the duration of abstinence is less than 48 hours, sperm concentration
may be reduced, but motility will probably not be affected.

2. Two semen samples should be collected for initial evaluation. The interval of time
between the collections will depend on local circumstances but should not be less than
7 days or more than 3 months apart. If the results of these assessments are remarkably
different, additional semen samples should be tested because marked variations in
sperm output may occur within the same individual. Analysis of multiple semen
specimens provides a reliable screen in the evaluation of male factor infertility.
Information and support are important since semen analysis cause a moderate amount
of stress to the individual.

3. Ideally the sample should be collected in the privacy of a room near the laboratory.
If not, it should be delivered to the laboratory within 1hour after collection.

4. The sample should be obtained by masturbation and ejaculated into a clean, wide-
mouthed glass or plastic container. If plastic is used, it should be checked for lack of
toxic effects on spermatozoa. The container should be warm to minimize the risk of
cold shock. Ordinary condoms must not be used for semen collection because they
may interfere with the viability of spermatozoa. In cases in which masturbation is not
possible or against an individual’s values, the specimen can be collected in a non-
spermicidal condom following intercourse. It has been shown that semen samples
collected during intercourse using a special plastic condom or a silastic collection
device tend to have better parameters. Other authors, referring to their experience, hold
the view that the quality of the specimen when collected in this way is generally
compromised. This way of collection should be considered for a second sample if the
first one shows a relatively low volume. Coitus interruptus is not acceptable as a
means of collection because it is possible that the first portion of the ejaculate, which
contains the highest concentration of spermatozoa, will be lost. Moreover, there will be
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cellular and bacteriological contamination of the sample and the acid pH of the vaginal
fluid will adversely affect sperm motility.

5. Incomplete samples should not be analyzed, particularly if the first portion of the
ejaculate is lost. The sample should be protected from extremes of temperature (not
less than 20°C and not more than 40°C) during transport to the laboratory. The sample
should be examined immediately after liquefaction and certainly within 1 hour of
ejaculation.

Laboratory technicians should be aware that semen samples may contain harmful
viruses (such as HIV and viruses causing hepatitis and herpes) and should therefore
be handled with due care.
Macroscopic evaluation

Appearance
The semen sample is first evaluated by simple inspection. A normal sample has a grey-
opalescent appearance, is homogenous and liquefies within 60 minutes at room
temperature under the influence of enzymes of prostatic origin. In some cases,
liquefaction does not occur within the normal time period and this fact should be
recorded, as it may suggest functional disturbance of the prostate. Normal semen
samples may contain jelly-like grains which do not liquefy.
The sample may appear clear if the sperm concentration is too low. It may also appear
brown when red blood cells are present in the ejaculate (haematospermia).
The presence of mucous streaks may interfere with the counting procedure and
suggests inflammation or abnormal liquefaction.
Samples which do not liquefy need additional treatment such as exposure to bromelin,
to make the sample amenable to analysis.
The sample should be well mixed in the original container. Incomplete mixing is
probably a major contributor to errors in determining sperm concentration.

Consistency
The consistency, also called viscosity, of the liquefied sample can be estimated by
gentle aspiration into a 5-ml pipette and then allowing the semen to drop by gravity
and observing the length of the thread formed. A normal sample leaves the needle as
small discrete drops, while in cases of abnormal consistency the drop will form a
thread of more than 2 centimetres. Another method to estimate consistency does not
use needles and is performed by introducing a glass rod into the sample and observing

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the thread that forms on withdrawal of the rod. Again the thread should not exceed 2
centimetres.
Increased consistency has the same clinical meaning as abnormal liquefaction, and
may be related to prostate dysfunction resulting from chronic inflammation.
Very viscous specimens can impair the availability of fertile sperm at the site of
fertilization.

Volume
The major component of the ejaculate volume is made up of secretions from the
accessory glands. The bulk of the volume is secreted by the seminal vesicles and
between 0.5 and 1 millilitres originates from the prostate. The volume of the ejaculate
should be measured either with a graduated cylinder or by aspirating the whole sample
into a wide-mouthed pipette by means of a mechanical device. The sample volume can
also be determined directly in the collection tube by weighing; assuming 1millitres
equals to 1gram. Thereby, loss of volume associated with transfer from the collection
tube to either another tube or a pipette can be avoided.
A low ejaculate volume can reflect abnormalities in accessory sex gland fluid synthesis
or secretion. It can also be indicative of a physical obstruction somewhere in the
reproductive tract, or may occur in cases of incomplete or (partially) retrograde
ejaculation.
Large volumes are sometimes found in association with varicocele or after relatively
long periods of sexual abstinence.

pH
The pH is determined by acidic secretions of the prostate and alkaline secretions of the
seminal vesicles. It should normally be in the range of 7.2-8.0.
To test pH, pH litmus paper range 6.1 to 10.0 is used. Whatever type of pH paper is
used for this analysis, its accuracy should be checked against known standards before
the use in routine semen analysis.
If the pH exceeds 8.0, infection should be suspected with decreased secretion of acidic
products by the prostate, such as citric acid. Abnormal pH may also be recorded in
cases of incomplete ejaculation. Extremely acidic pH (<6.5) is found in cases of
agenesis (or occlusion) of the seminal vesicles.

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Initial microscopic investigation

During the initial microscopic investigation of the sample, estimation of motility and
concentration of spermatozoa is performed. The presence of cells other than
spermatozoa and of agglutination of spermatozoa is determined.

Motility
In recent years, a number of techniques for objective assessment of movement
characteristics of human spermatozoa have been introduced by using computer-
assisted semen analysis (CASA) systems. For the purpose of conventional analysis, a
simple classification system which provides the best possible assessment of sperm
motility without resorting to complex equipment is recommended.
A fixed volume of semen is delivered onto a clean glass slide and covered with a
22x22 millimetre glass coverslip. It is important that the volume of semen and the
dimension of the coverslip are standardized so that the analyses are always carried out
in a preparation with fixed depth. This depth allows full expression of the rotating
movement of normal spermatozoa. The preparation is then examined at a
magnification of x400-600. An ordinary light microscope can be used for unstained
preparations, particularly if the condenser is lowered to disperse the light. However, a
phase-contrast microscope is preferable.
The weight of the coverslip spreads the sample for optimal viewing. The freshly made,
wet preparation is left to stabilize for approximately one minute. Motility estimation
can conveniently be carried out at a room temperature between 18 and 24°C. At
temperatures outside this range, some alteration in sperm motility will occur and this
must be standardized in the laboratory.
The microscopic field is scanned systematically and the motility of each spermatozoon
encountered is graded a, b ,c or d according to whether it shows:
(a) Rapid progressive motility.
(b) Slow or sluggish progressive motility
(c) Non-progressive motility.
(d) Immotility.
Spermatozoa graded (a) are supposed to display rapid progressive motility along a
linear track, covering a distance of at least 20 microns (half the length of a
spermatozoon) per second.

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At least 100 spermatozoa are classified in this way. Visual field close to the border of
the coverslip should be avoided.
It is advisable to repeat the procedure on a second drop of semen processed in the same
way.

Estimation of sperm concentration

The concentration can be estimated roughly during the initial examination in order to
determine the dilution procedure to be used and to indicate whether centrifugation may
be required to prepare an adequate smear for morphologic analysis.

Cells other than spermatozoa

The ejaculate usually contains cells other than spermatozoa. These include polygonal
cells from the urethral tract. If many of these are present, and they are covered with
bacteria then it is probably that the sample was obtained by coitus interruptus and the
cells originate from the vagina. Spermatogenic cells and white blood cells (WBC),
which are often referred to as "round cells", are present in almost every semen sample.
By conventional light microscopy or sperm staining techniques it is not possible to
reliably differentiate WBC from immature germ cells in semen. In contrast, the
cytochemical Peroxidase method reliably identifies granulocytes, the most prevalent
WBC type in semen. The method is cheap, fast and easy to perform. The gold standard
for the detection of all WBC populations in semen is immuno-cytology using
monoclonal antibodies. However, it is expensive and time-consuming, thus remaining
a research tool at present. For clinical purposes, the Peroxidase method is ideally
suited to detect granulocytes.
The method aims at the counting of Peroxidase-positive round cells in a
haemocytometer. The working solution is prepared by combining 1millillitre of
saturated NH4Cl solution, 1milillitre of 5% of sodium citrate solution, 9ml of ortho-
toluidine solution and 1 drop of H 2O2. This solution is mixed before use and can be
conserved for 24 hours after preparation. The procedure consists of mixing 0.1millitre
of semen with 0.9millilitre of the working solution to achieve a total volume of
1millilitre. This mixture is shaken for 2 minutes. It is then left for 20-30 minutes at
room temperature and mixed again by shaking. The mixture is now transferred onto a
haemocytometer chamber for leukocytes and the number of Peroxidase-positive cells
which stain brown is counted. Peroxidase-negative cells remain unstained and are
counted in the haemocytometer chamber. The differentiation of round cells into either

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Peroxidase-positive polymorphonuclear granulocytes or Peroxidase-negative
Spermatogenic cells or lymphocytes is of clinical relevance. The presence of an
excessive number of Peroxidase-negative mostly Spermatogenic cells suggests
pathology at the level of the seminiferous epithelium with inadequate spermatogenesis
and premature release of spermatids spermatocytes or, rarely, spermatogonia. The
pathological meaning of the presence of an elevated number of WBC is still a matter of
dispute. Some reports have demonstrated that leukocytospermia appears to be of no
diagnostic value to identify men with actual microbial infections. Also, measurement
of seminal leukocytes in routine semen analysis appears to be of little prognostic value
with regard to male fertilizing potential.

Agglutination
Agglutination of spermatozoa means that motile spermatozoa stick to each other, head
to head, midpiece to midpiece, tail to tail, or mixed, like midpiece to tail. The
adherence of either immotile or motile spermatozoa to mucus threads, to cells other
than spermatozoa, or to debris is not considered agglutination and should not be
recorded as such.
The presence of agglutination is suggestive of, but not sufficient evidence to prove the
existence of an immunological factor of fertility. The extent of agglutination may be
important but even the presence of only a few groups of small numbers of agglutinated
spermatozoa should be recorded. In case of agglutination, sperm culture must be
performed in order to exclude infection with of bacteria such as Escheria coli. Sperm
agglutination could be used also as indication for anti-sperm antibody testing of
infertile men.

Further microscopic examination

Sperm viability
Vital staining of the spermatozoa allows quantification of the fraction of living cells
independently of their motility. Live and dead sperm are distinguished by adding one
drop of eosin Y stain to one drop of semen at room temperature (one to two minutes)
and smearing the mixture on a microscopic slide. 100 spermatozoa are classified as
coloured orange-red, if the stain has passed through the membrane and therefore the
cell is considered dead, or non-stained, the cell then being considered alive.
This staining technique makes it possible to differentiate spermatozoa that are
immotile but alive from those that are dead. Reduced percentage of motility with a
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high percentage of viable sperm may reflect structural or metabolic abnormalities of
sperm that are derived from abnormalities in testicular function or anti motility factors
in the seminal plasma.
This technique also provides a check on the accuracy of the motility evaluation, since
the percentage of dead cells should not exceed the percentage of immotile
spermatozoa.

Hypo-osmotic swelling (HOS) test

The hypo-osmotic swelling (HOS) test measures sperm membrane integrity by
examining its ability to swell when exposed to hypo-osmotic media, and has been
claimed to be relevant to fertilizing ability. The rationale of the test is based on the
assumption that an undamaged sperm tail membrane permits passage of fluid into the
cytoplasmic space causing swelling and the pressure generated leads to curling of tail
fibres, while the damaged or chemically inactive membrane allows fluid to pass across
the membrane without any accumulation and accordingly no cytoplasmic swelling and
curling of the tail occur.
The HOS test should not be used as a sperm function test but may be used as an
optional, additional vitality test. It is simple to perform and easy to score and gives
additional information on the integrity and the compliance of the cell membrane of the
sperm tail.

Counting the spermatozoa

The concentration of spermatozoa should be determined using the haemocytometer
method.
In this procedure a 1:20 dilution from each well-mixed sample is prepared by diluting
liquefied semen with a diluent. The latter is prepared by adding 50 grams of sodium
carbonate (NaHCO3), 10mllilitres of 35% (v/v) formalin and, optionally, 0.25 grams of
Tryphan blue or 5millilitres of saturated aqueous gentian violet to distilled water and
making up the solution to a final volume of 1000ml. The stain needs not to be included
if a phase-contrast microscopy is used. If the preliminary examination of the semen
indicates that the concentration of spermatozoa present is either excessively high or
low, then the extent to which the sample is diluted should be adjusted accordingly. For
samples containing less than 20x10 6 spermatozoa/ml, a 1:10 dilution should be used;
6
for samples containing more than 100x10 spermatozoa/ml, a 1:50 dilution may be
appropriate. Both chambers of the haemocytometer are scored and the average count is

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calculated, provided that the difference between the two counts does not exceed 1/20
of their sum (less than 10% difference). If the two counts are not within 10%, they are
discarded, the sample dilution re-mixed and another haemocytometer prepared and
counted.
The total number of spermatozoa per ejaculate reflects spermatogenesis and is related
to the duration of sexual abstinence.
Perhaps the most widely utilized semen parameter is sperm count. Men with <20x10 6
spermatozoa per ml are typically deemed sub-fertile, and men with counts <5x10 6
spermatozoa/ml are often considered infertile.
Other authors have confirmed that in patients with sperm counts <20x10 6/ml the
fertility potential is significantly impaired .However, it must be emphasized that
patients with sperm counts <20x106 per ml are not infertile. It simply takes them a
substantially longer period of time to achieve pregnancies.

Analysis of the morphological characteristics of spermatozoa

Sperm cells represent a unique population in which up to 50% (up to 70% according to
WHO criteria 1992 and up to 86% according to strict criteria) of the cells can have
morphological defects in normal fertile individuals. Although the morphological
variability of the human spermatozoon makes differential sperm morphology
evaluation very difficult, observations on the selection of spermatozoa recovered from
the female reproductive tract (especially in post coital cervical mucus) helped to define
the appearance of a normal spermatozoon. The normal head should be oval in shape.
Allowing for the slight shrinkage that fixation and staining induce; the length of the
head should be 4.0-5 microns the width 2.5-3.5 microns. The length-to-width ratio
should be 1.50 to 1.75 microns. There should be a well-defined acrosomal region
comprising 40-70% of the head area. There must be no neck, midpiece or tail defects
and no cytoplasmic droplet more than one-third the size of a normal sperm head. This
classification scheme requires that all borderline forms be considered abnormal.
The following categories of defects should be scored.
 Head shape/size defects, including large, small, tapering, pyriform, amorphous,
vacuolated (>20 of the head area occupied by unstained vacuolar areas), or
double heads, or any combination of these.
 Neck and midpiece defects, including absent tail, non inserted or bent tail (the
tail forms an angle of about 90° to the long axis of the head),

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 distended/irregular/bent midpiece, abnormally thin midpiece or any
combination of these.
 Tail defects, including short, multiple, hairpin, broken, irregular width, or
coiled tails, tails with terminal droplets, or any combination of these.
 Cytoplasmic droplets greater than one-third of the area of a normal sperm head.
The traditional feathering technique (whereby the edge of a second slide is used to
drag a drop of semen along the surface of the cleaned slide) may be used to make thin
smears of spermatozoa. The Papanicolaou smear for staining of spermatozoa is the
method most widely used in andrology laboratories. Sperm morphology gives
information for the function of the reproductive tract and is a predictor of man’s
fertility potential.
Physical sperm aberrations may occur during the production of sperm or during
storage in the epididymis. In cases of Teratozoospermia, one should start first by
excluding the presence of monomorphic genetic syndromes such as globozoospermia,
microcephaly and short tail spermatozoa. The increased number of immature
spermatozoa may be due to epididymal dysfunction or is a consequence of frequent
ejaculations. The increased numbers of spermatozoa with tapering heads are found in
association with varicocele.
The usefulness of sperm morphology assessment as a predictor of a man’s fertilizing
potential has often been challenged due to different classification systems, various
slide preparation techniques and problems with reproducibility because of observer
variations.

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(Stained human Spermatozoa)

Testing for antibody coating of spermatozoa

The presence of anti-sperm antibodies in semen can alter the fertilizing ability of the
spermatozoa. Being haploid, sperm cells are immunogenic and display different
surface antigens from their diploid counterparts. Under normal circumstances, they are
protected from the man’s immune system by a basal membrane constituting the blood-
testis barrier. When this barrier is ruptured, sperm cells induce the synthesis of anti-
sperm antibodies. The presence of sperm antibodies coating the spermatozoa is typical
of and is considered to be specific for immunologic infertility. Sperm antibodies in
semen belong to the immunoglobulin classes IgG, IgA or rarely IgM. There are some
data suggesting that IgA antibodies may have greater clinical importance than IgG
antibodies. The screening test for antibodies is performed on the fresh semen sample
and makes use of either the Immunobead method or the mixed antiglobulin reaction
test (MAR test).

Immunobead test

Immunobeads are polyacrylamide spheres with co-valent bound rabbit anti-human

immunoglobulins. The presence of IgG, IgA and IgM antibodies can be assessed
simultaneously with this test. Spermatozoa are washed of seminal fluid by repeated
centrifugation and re-suspended in buffer. The sperm suspension is mixed with a
suspension of Immunobeads. The test is considered positive when 25% or more of
motile spermatozoa have Immunobead binding.

MAR test
The IgG MAR test is performed by mixing fresh, untreated semen with latex particles
or sheep blood cells coated with human IgG. A monospecific antihuman-IgG
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antiserum is added to this mixture. The formation of mixed agglutinates between
particles and motile spermatozoa proves the presence of IgG antibodies on the
spermatozoa. The diagnosis of immunologic infertility is probable when 50% or more
of the motile spermatozoa have particles adherent. Immunologic infertility is suspected
when 10%-50% of the motile spermatozoa have adherent particles.
Sperm antibodies could influence sperm function in a variety of ways. For example,
sperm agglutination and immobilizing antibodies might limit the number of fertile
sperm cells at the site of fertilization. Antibody production against sperm surface
macromolecules could interfere with critical physiologic fertilization precursor events,
such as capacitation and the acrosome reaction. It is also possible that antibodies
produced against essential intra-acrosomal enzyme systems, such as proacrosin-acrosin
could impair sperm penetration through egg investment.
Whatever the method of action, sperm antibodies have been shown to impair fertility
and may account for up to 10% of couples whose infertility is unexplained.
Infection of the genital tract, varicocele, cryptorchidism, testicular torsion and auto-
immune disease are the most frequent conditions associated with anti-sperm antibodies
Biochemical analysis
There are various biochemical markers of accessory gland function, like citric acid,
zinc and acid phosphatase for the prostate gland; fructose and prostaglandins for the
seminal vesicles, free L-carnitine, glycerophosphocoline, and alfa-glucosidase for the
epididymis. A low secretory function is reflected in a low total output of the specific
marker(s), which may therefore be used for the assessment of accessory gland
secretory function. An infection can sometimes cause a considerable decrease in the
secretory function of these glands. Fructose determination is also useful in cases of
dysgenesis of seminal vesicles and in the rare cases of ejaculatory duct obstruction.

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 Chapter Fifteen:

Mausoleum Techniques:
Learning Outcomes:
 At the end of the instruction the learner should be able to:
 Describe death
 Define embalming terms
 Describe historical development of embalming
 State the importance of embalming dead bodies
 Name embalming fluids in common use
 Explain the importance of cultural and religious rights to the dead
 Describe the embalming process
Death:
Death is the cessation of all biological functions that sustain a living organism. It is a
phenomena which commonly bring about death. It is caused by various factors that
include: Biological aging (Senescence), predation, malnutrition, disease, suicide,
murder and accidents or trauma resulting in internal injury.
Bodies of living organisms begin to decompose shortly after death.
Signs of death or strong indications that an animal is no longer alive are:
 Cessation of breathing
 Cardiac arrest (no pulse)
 Pallor mortis, paleness which happens in 15- 20 minutes after death
 Algor mortis, reduction of body temperature after death
 Livor mortis, settling of blood in the lower portion of the body depending on
the position at the time of death
 Rigor mortis, the limbs of the corpse become stiff and difficult to move or
manipulate
 Decomposition, reduction into smaller forms of matter accompanied by strong
unpleasant odour
Once death is confirmed there is issuance of legal death certificate in case of human
beings.

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Embalming
Embalming, in most modern cultures, is the art and science of temporarily preserving
human remains to forestall decomposition and to make them suitable for public display
at a funeral. The three goals of embalming are thus sanitization, presentation and
preservation (or restoration) of a corpse to achieve this effect. Embalming has a very
long and cross-cultural history, with many cultures giving the embalming processes a
greater religious meaning.

History

Embalming has been practiced in many cultures. In classical antiquity, perhaps the
ancient culture that had developed embalming to the greatest extent was that of ancient
Egypt, which developed the process of mummification. They believed that preservation
of the mummy empowered the soul after death, which would return to the preserved
corpse. Other cultures that had developed embalming processes include the Incas and
other cultures of Peru, whose climate also favoured a form of mummification.
However some of the best preserved bodies in the world are from Han dynasty China.
It was thought that a special liquid in which the bodies were embedded (solutions
containing mercury and antimony salts amongst others), may have been of a certain
influence. The actual cause of the preservation-which started declining rapidly once
the bodies were unearthed-was the very exceptional low temperature conditions
obtained at the depths at which the tombs were located, under several layers of
charcoal and clay, permitting ideal temperatures and humidity levels which were
maintained throughout the seasons for centuries.
These mummies are nowadays stored in special refrigerated chambers which simulate
the original conditions in which they were discovered to prevent further acceleration of
putrefaction .
Embalming in Europe had a much more sporadic existence. It was attempted from time
to time, especially during the Crusades, when crusading noblemen wished to have their
bodies preserved for burial closer to home. Embalming began to come back into
practice in parallel with the anatomists of the Renaissance who needed to be able to
preserve their specimens. Arterial embalming is believed to have been first practiced in
the Netherlands in the 17th century by Frederik Ruysch but his liquor balsamicum
preservative was kept a secret to the grave and his methods were not widely copied.

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Contemporary embalming methods advanced markedly during the American Civil
War, which once again involved many servicemen dying far from home, and their
family wishing them returned for local burial. Dr. Thomas Holmes received a
commission from the Army Medical Corps to embalm the corpses of dead Union
officers to return to their families. Military authorities also permitted private
embalmers to work in military-controlled areas. The passage of Abraham Lincoln's
body home for burial was made possible by embalming and it brought the possibilities
and potential of embalming to a wider public notice.
In 1867, the German chemist August Wilhelm von Hofmann discovered formaldehyde,
whose preservative properties were soon discovered and which became the foundation
for modern methods of embalming, replacing previous methods based on alcohol and
the use of arsenical salts.
In the 19th and early 20th centuries arsenic was frequently used as an embalming fluid
but has since been supplanted by other more effective and less toxic chemicals. There
were questions about the possibility of arsenic from embalmed bodies later
contaminating ground water supplies. There were also legal concerns as people
suspected of murder by arsenic poisoning could claim that the levels of poison in the
deceased's body were a result of embalming post mortem rather than evidence of
homicide.
Embalming is distinct from taxidermy. Embalming preserves the human body intact,
whereas taxidermy is the recreation of an animal's form often using only the creature's
skin mounted on an anatomical form.
Modern embalming is most often performed to ensure a better presentation of the
deceased for viewing by friends and relatives - as everything else being equal, an
embalmed body will look better than one that is unembalmed and putrefying. A
successful viewing of the cadaver is considered by many credible authorities to be
helpful in the grieving process. It allows the mourners to form a memory picture of the
deceased. Embalming is also a general legal requirement for international repatriation
of human remains (although exceptions do occur) and by a variety of laws depending
on locality, such as for extended time between death and final disposition or above
ground entombment.

Terms for embalmers

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The roles of a Funeral Director and an embalmer are different. A funeral director is a
person who arranges for the final disposition of the deceased and who may or may not
prepare (including embalming) the deceased for viewing (or other legal requirements).
An embalmer is someone who has been trained in the art and science of embalming
and may not have any contact with the family, although many people fill both roles.
The term mortician is becoming outdated, but may refer either to a funeral director or
to an embalmer, or both. Embalming training commonly involves formal study in
anatomy, thanatology, chemistry, and specific embalming theory (to widely varying
levels depending on the region of the world one lives in) combined with practical
instruction in a mortuary with a resultant formal qualification granted after the passing
of a final practical examination and acceptance into a recognized society of
professional embalmers.
Legal requirements over who can practice vary geographically; some regions or
countries have no specific requirements. Additionally, in many places, embalming is
not done by trained embalmers, but rather by doctors who, while they have the
required anatomical knowledge, are not trained specialists in this field. Today,
embalming is common practice in North America and New Zealand while it is
somewhat less frequent in Europe. In some countries, permits or licenses are required;
in others it is performed only by medical practitioners, and the costs can be relatively
high. In the United States, the title of an embalmer is based largely on the state that
they are licensed in. In Virginia, Maryland, a funeral director is someone who is
licensed only to make arrangements and handle the business side of the funeral home,
while a mortician is licensed to do these things as well as to embalm.

Modern practices

Instruments used for embalming

As practiced in the funeral homes of the Western World (notably North America),
embalming uses several steps. Modern embalming techniques are not the result of a
single practitioner, but rather the accumulation of many decades, even centuries, of
research, trial and error, and invention. A standardized version follows below, but
variation on techniques is very common.

The deceased is placed on the mortuary table in the supine anatomical position with
the head elevated by a head block. The first step in embalming is obviously to check

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that the individual is in fact deceased, and then verify the identity of the body
(normally via wrist or leg tags). At this point embalmers commonly perform basic tests
for signs of death, noting things such as clouded-over corneas, lividity, and rigor
mortis or by simply attempting to palpate a pulse in the carotid or radial artery. In
modern times people awakening on the preparation table is largely the province of
horror fiction and urban myth.

Any clothing on the corpse is removed and set aside and any personal effect such as
jewelry is inventoried. A modesty cloth is sometimes placed over the genitalia. The
corpse is washed in disinfectant and germicidal solutions. During this process the
embalmer bends, flexes and massages the arms and legs to relieve rigor mortis. The
eyes are posed using an eye cap that keeps them shut and in the proper expression. The
mouth may be closed via suturing with a needle and ligature, using an adhesive, or by
setting a wire into the maxilla and mandible with a needle injector, a specialized device
most commonly utilized in North America and unique to mortuary practice. Care is
taken to make the expression look as relaxed and natural as possible and ideally a
recent photograph of the deceased while still living is used as a template. The process
of closing the mouth, eyes, shaving and other manipulations, is collectively known as
setting the features.

The actual embalming process usually involves four parts:

1. Arterial embalming, this involves the injection of embalming chemicals into the
blood vessels, usually via the right common carotid artery. Blood and interstitial fluids
are displaced by this injection and, along with excess arterial solution, are expelled
from the right jugular vein and collectively referred to as drainage. The embalming
solution is injected with a centrifugal pump and the embalmer massages the body to
break up circulatory clots as to ensure the proper distribution of the embalming fluid.
This process of raising vessels with injection and drainage from a solitary location is
known as a single-point injection. In cases of poor circulation of the arterial solution
additional injection points (commonly the axillary, brachial or femoral arteries, with
the ulna, radial and tibial vessels if necessary) are used. The corresponding veins are
commonly also raised and utilized for the purpose of drainage. Cases where more than
one vessel is raised are referred to as multiple-point injection, with a reference to the
number of vessels raised (like a six-point injection or six-pointer). An injection
utilizing both the left and right carotids is specifically referred to as a restricted
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cervical injection (RCI), while draining from a different site to injection ( injecting
arterial fluid into the right common carotid artery and draining from the right femoral
vein) is referred to as a split (or sometimes cut) injection.

2. Cavity embalming refers to the replacement of internal fluids inside body cavities
with embalming chemicals via the use of an aspirator and trocar. The embalmer makes
a small incision just above the navel (two inches superior and two inches to the right)
and pushes the trocar in the chest and stomach cavities to puncture the hollow organs
and aspirate their contents. He/she then fills the cavities with concentrated chemicals
that contain formaldehyde. The incision is either sutured closed or a "trocar button" is
secured into place.

3. Hypodermic embalming is a supplemental method which refers to the injection of

embalming chemicals into tissue with a hypodermic needle and syringe, which is
generally used as needed on a case by case basis to treat areas where arterial fluid has
not been successfully distributed during the main arterial injection.

4. Surface embalming, another supplemental method, utilizes embalming chemical to

preserve and restore areas directly on the skins surface and other superficial areas as
well as areas of damage such as from accident, decomposition, cancerous growth or
skin donation.

A typical embalming takes several hours to complete. An embalming case that requires
more attention or has unexpected complications could take substantially longer. The
repair of an autopsy case and the restoration of a long-bone donor are two such
examples.

Grooming

Restoration tools, Museum of Funeral Customs

After the body is rewashed and dried, a moisturizing cream is applied to the face. The
body will usually sit for as long as possible for observation by the embalmer. After
being dressed for visitation/funeral services, cosmetics are applied to make the body
appear more lifelike and to create a "memory picture" for the deceased's friends and
relatives. For babies who have died, the embalmer may apply a light cosmetic massage
cream after embalming to provide a natural appearance; massage cream is also used on

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the lips to prevent them from dehydrating, and the infant's mouth is often left open a
bit for a more natural expression. If possible, the funeral director uses a light,
translucent cosmetic; sometimes, heavier, opaque cosmetics are used to hide bruises,
cuts, or discoloured areas. Makeup is applied to the lips to mimic their natural colour.
Sometimes a very pale or light pink lipstick is applied on males, while brighter
coloured lipstick is applied to females. Hair gels or baby oil is applied to style the hair
of males; while hairspray is applied to style the hair of females. Powders (especially
baby powder) are applied to the body to eliminate odours, and it is also applied to the
face to achieve a Matte and Fresh Effect to prevent oiliness of the corpse. Mortuary
cosmetizing is not done for the same reason as make-up for living people; rather, it is
designed to add depth and dimension to a person's features that lack of blood
circulation has removed. Warm areas - where blood vessels in living people are
superficial, such as the cheeks, chin, and knuckles - have subtle reds added to recreate
this effect, while browns are added to the palpabrae (eyelids) to add depth, especially
important as viewing in a casket creates an unusual perspective rarely seen in everyday
life. During the viewing, pink-colored lighting is sometimes used near the body to lend
a warmer tone to the deceased's complexion.
A photograph of the deceased in good health is often sought in order to guide the
embalmer's hand in restoring the body to a more lifelike appearance. Blemishes and
discolorations (such as bruises, in which the discoloration is not in the circulatory
system and cannot be removed by arterial injection) occasioned by the last illness, the
settling of blood, or the embalming process itself are also dealt with at this time
(although some embalmers utilize hypodermic bleaching agents, such as phenol based
cauterants, during injection to lighten discoloration and allow for easier cosmetizing).

Clothing

In the western world, men are typically buried in business attire, such as a suit or coat
and tie, and women in semi-formal dresses or pant suits. In recent years, a change has
occurred and many individuals are now buried in less formal clothing, such as what
they would have worn on a daily basis, or other favorite attire. Clothing worn can also
reflect the deceased person's profession or vocation: Priests and ministers are often
dressed in their liturgical vestments, and military and law enforcement personnel often
wear their uniform. Underwear, singlets, bras, briefs and hosiery are all used if the
family so desires, and the deceased is dressed in them as they would be in life.

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In certain instances a funeral director will request a specific style of clothing, such as a
collared shirt or blouse, in order to cover traumatic marks or autopsy incisions. In other
cases clothing may be cut down the back and placed on the deceased from the front to
ensure a proper fit. In many areas of Asia and Europe, the custom of dressing the body
in a specially designed shroud/funeral gown, rather than in clothing used by the living,
is preferred.

After the deceased has been dressed, they are placed in the casket (the term casket is
derived from older usage to refer to a "jewel box", it is called a coffin when the
container is anthropoid (a stretched hexagon form) for the various funeral rites. It is
common for photographs, notes, cards and favourite personal items to be placed in the
casket with the deceased. Even bulky and expensive items, such as electric guitars, are
occasionally interred with a body. In some ways this mirrors the ancient practice of
placing grave goods with a person for use/enjoyment in the afterlife. In traditional
Chinese culture, paper substitutes of the goods are buried or cremated with the
deceased instead, as well as paper money specifically purchased for the occasion.

Embalming chemicals

Embalming chemicals are a variety of preservatives, sanitizers, disinfectant agents and

additives used in modern embalming to temporarily delay decomposition and restore a
natural appearance for viewing a body after death. A mixture of these chemicals is
known as embalming fluid and is used to preserve deceased individuals, sometimes
only until the funeral, other times indefinitely.
Typical embalming fluid contains a mixture of formaldehyde, glutaraldehyde, ethanol,
humectants, and wetting agents and other solvents. The formaldehyde content
generally ranges from 5 to 35 percent and the ethanol content may range from 9 to 56
percent.
Specialist embalming

Badly decomposing bodies, trauma cases, frozen and drowned bodies, and those to be
transported for long distances also require special treatment beyond that for the
"normal" case. The restoration of bodies and features damaged by accident or disease
is commonly called restorative art or demisurgery and all qualified embalmers have
some degree of training and practice in it. For such cases, the benefit of embalming is
startlingly apparent. In contrast though, many people have unreasonable expectations

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of what a dead body should look like, due to the unrealistic portrayal of "dead" bodies
(usually by live actors) in movies and television shows. Viewers generally have an
unrealistic expectation that a body going through decomposition should look as it did
before death. Ironically, the work of a skilled embalmer often results in the deceased
appearing natural enough that the embalmer appears to have done nothing at all.
Normally cosmeticians are very happy when someone can bring in a picture and the
decedent's regular makeup, if worn, to help make their loved one to look as they did
when alive.

Embalming autopsy cases differs from standard embalming because the nature of the
post-mortem examination irrevocably disrupts the circulatory system, due to the
removal of the organs and viscera. In these cases, a six-point injection is made through
the two iliac or femoral arteries, subclavian or axillary vessels, and common carotids,
with the viscera treated separately with cavity fluid or a special embalming powder in
a viscera bag. Long-term preservation requires different techniques, such as using
stronger preservative chemicals and multiple injection sites to ensure thorough
saturation of body tissues.
Embalming is meant to temporarily preserve the body of a deceased person.
Regardless of whether embalming is performed, the type of burial or entombment, and
the materials used- such as wood or metal caskets and vaults- the body of the deceased
will eventually decompose. Modern embalming is done to delay decomposition so that
funeral services may take place or for the purpose of shipping the remains to a distant
place for disposition.
Embalming for anatomy education
A rather different process is used for cadavers embalmed for dissection by doctors and
medical students. Here, the first priority is for long term preservation, not presentation.
As such, medical embalmers use embalming fluids that contain concentrated
formaldehyde (37–40%, known as formalin)/glutaraldehyde as well as phenol and are
made without dyes or perfumes. Many embalming chemical companies make
specialized anatomical embalming fluids.
Anatomical embalming is performed into a closed circulatory system. The fluid is
usually injected with an embalming machine into an artery under high pressure and
flow and allowed to swell and saturate the tissues. After the deceased is left to sit for a
number of hours, the venous system is generally opened and the fluid allowed to drain
out, although many anatomical embalmers do not use any drainage technique.

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Anatomical embalmers may choose to use gravity-feed embalming, where the
container dispensing the embalming fluid is elevated above the body's level and fluid
is slowly introduced over an extended time, sometimes as long as several days. Unlike
standard arterial embalming, no drainage occurs and the body distends extensively
with fluid. The distension eventually reduces, often under extended (up to six months)
refrigeration, leaving a fairly normal appearance. There is no separate cavity treatment
of the internal organs. Anatomically embalmed cadavers have a typically uniform grey
colouration, due both to the high formaldehyde concentration mixed with the blood
and to the lack of red colouration. Formaldehyde mixed with blood causes the grey
discoloration also known as "formaldehyde grey" or "embalmers grey" (added
normally to standard, non-medical, embalming fluid)

Religious practices

There is much difference of opinion amongst different faiths as to the permissibility of

embalming. A brief overview of some of the larger faiths positions are examined
below.

 Neopagans generally discourage embalming, believing it unnatural to disrupt the

physical recycling of the body to the Earth. They encourage the use of green
graveyards, where the body is placed in a biodegradable casket and buried under a
tree instead of a tombstone.

 Most branches of the Christian faith generally allow embalming. Some bodies
within Eastern Orthodoxy profess an absolute ban against embalming except when
required by law or other necessity, while others may discourage but do not prohibit
it. In general the decision on embalming is one that is dictated by the personal
preference of the family rather than a specific church policy.

 The Church of Jesus Christ of Latter-day Saints does not discourage or prohibit
embalming. Often, due to the custom of church members dressing the deceased,
embalming is given preference.

 Members of Iglesia ni Cristo allow embalming for the view of their loved ones. It
forbids autopsy and cremation because they believe the body of the deceased is
sacred and should be cared for with respect. They dress and groom the deceased as

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 they looked in life. The preferred method is arterial embalming, in which
formaldehyde is injected into the body.

 Many authorities hold that Hinduism does not accept embalming. In practice, this
is not an adamant prohibition, and embalming for those of Hindu faith is known to
occur, generally for repatriation to India or the South Pacific and for the purposes
of viewing and funerary rites at the family home prior to final cremation.
Traditionally, a dead body should be cremated before sunset, and embalming is
neither common nor widespread.

 Members of the Bahá'í Faith are not embalmed. Instead, the body is washed and
placed in a cotton, linen or silk shroud. The body is to be buried within one hour's
journey from the place of death, if this is feasible. Cremation is also forbidden.

 Zoroastrians traditionally hold a type of sky burial within a structure known as a

Tower of Silence in which the body is exposed to weathering and predation to
dispose of the remains, and thus embalming the body is contrary to their funeral
designs. This is due to the Zoroastrian belief that the dead body is unclean and the
pure elements of earth and fire should not be allowed to come into contact with it.
This practice is not universally performed any more, and many Iranian
Zoroastrians perform traditional cremations and burials instead.

 Traditional Jewish law forbids embalming, and burial is to be done as soon as

possible - preferably within 24 hours. However, under certain circumstances,
burial may be delayed if it is impossible to bury a person immediately, or to permit
the deceased to be buried in Israel. Guidance of a Rabbi or the local chevra kadisha
(Jewish Burial Society) should be sought regarding any questions, as particular
circumstances may justify leniencies. Notably, the Biblical Joseph was, according
to the (Genesis 50:26) embalmed in the Egyptian fashion as was his father Israel
(Jacob) (Genesis 50:2).

 Muslims are required to be buried within 24 hours of death, if possible. Embalming

is forbidden. The body is washed and prepared specifically for interment. This
procedure is to be done according to the last will of the deceased, usually by a
close relative of the deceased who is of the same gender. He or she is then dressed
in a plain white burial shroud (for women, the hair, ears and neck are covered as

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 they were in life, preserving her dignity before men who are not closely related;
men are buried in their ihram clothing, or pilgrim garb, as worn during the Hajj in
Mecca). Muslims believe that the spirit remains with the body from death until
after burial, which is the reason for same-day burial, as well as the aforementioned
procedures; the body is treated with the same care and respect as in life so as to not
cause undue stress to the deceased. For the same reason, cremation is also
forbidden. Prayers and readings of the Qur'an are spoken aloud to give comfort to
the deceased, and the body is not left alone even for a time following the burial,
during which the deceased is buried (preferably without a casket) on his or her
right side, facing Mecca.
 In Islamic countries, a plain white burial shroud called a kafans, consisting of 3
pieces of white cotton clothing, is used to wrap the body.

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References:
1. Bradbury S. (1973). Hewers Textbook of Histology for medical students, 9 th
edition, William Heinemann Medical Books Ltd. London
2. Bancroft. J. D. (2013). Bancroft’s Theory and practice of Hitological
Techniques, 7th edition, Churchil Livingstone.
3. Burkitt, H. G., Young B. and Heath, J. W. (1993). Wheaters Functional
Histology, a text and colour atlas, 3rd edition, Churchill Livingstone, Hong
Kong.
4. Carton J., Daly R. and Ramani P. (2007). Clinical Pathology, Oxford
University Press.
5. D.M.Richard (2005). The pap test, American Society for Clinical Pathology.
6. Frederick, L.G. and Clarence G. (1989). The Principles and Practice of
Embalming, 5th edition, Dallas, TX: Professional Training Schools Inc &
Robertline
7. Frederick. M., Robert G. (2000). Embalming: History, Theory and Practice, 3 rd
edition, McGraw-Hill/Appleton & Lange.
8. Garg. K., Bahi. I. & Kaul. M. (2010), Textbook of Histology Colour Atlas, 4th
edition, CBS Publishers & Distributors.
9. Gill W. G. (2013), Cytopreparation Principles and Practice: essentials in
cytopathology, Springer Science, New York.
10. Jorgenson K.F., Van de Sande J. H. and Lin C. C. (1978) Chromasoma, 68:
287-302.
11. htt://www.biology.arizona.edu.,The cell cycle and Mitosis Tutorial, Retrieved
18/11/2011
12. Junquiera J. C., Carnairo J. and Kelley R. O (1989) Basic Histology, 6th edition,
Appleton and Lange, California.
13. Leeson C. R., Leeson T. S. and Paparo A. A. (1981) Textbook of Histology, 5th
edition, W. B. Saunders Company, USA.
14. Levison A. D., Reid R., Burt D. A., Harrison J. D. and Fleming S. (2008)
Muir’s Textbook of Pathology, 14th edition, Edward Arnold Publishers Ltd,
London.
15. Siegel. M. S. (1993), The Male infertility investigation and the role of the
andorlogy Laboratory, Journal Reproductive Medicine, 38(5). Pg 317-334.
16. Sood. R. (2009), Medical Laboratory Technology: Methods and interpretation,
6th edition, Jaypee Brothers, Medical Publishers.
195 | P a g e
 17. Waugh A., & Grant A., (2007), Ross and Wilson Anatomy and physiology,
Churchill Livingstone.
18. Wikipedia, the free encyclopaedia, Haematoxyline.png, retrieved on 3/8/2011.
19. World Health Organization, (1992), WHO Laboratory Manual for examination
of human Semen and Semen Cervical Mucus interaction, 3rd edition, Cambridge
University Press, Cambridge.

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