INTRODUCTION

The term histology is derived from two Greek words; ‘histo’ meaning tissues
and ‘logos’ meaning science. Histology is therefore the microscopic study of
tissues. Cytology on the other hand deals with the detailed study of individual
cell and its internal components.
A tissue is a group of predominantly similar cells specialized in a common
direction and set apart for performance of a common function. Usually cells are
grouped into tissues and each tissue type is highly specialized to perform
specific function.
Knowledge of tissue structure and function is important in understanding how
individual cells are organized to form tissues, organs, organ systems, and the
complete organism.

TISSUE PROCESSING IN HISTOLOGY

To visualize the microstructure of any tissue under a light microscope, the
specimen has to go through a thorough protocol of tissue fixation, tissue
processing, sectioning and staining.
Tissue collection: Commonly tissue is obtained from, autopsy, surgical
procedures, experimental animals (rabbit, rats, mice, etc.) either perfused or
decapitated (Guideline of ethics are always observed in any experimental
study).
Fixation
The first step in preparation of a tissue or organ sample for histological
examination is fixation and its purpose is to preserve structure. Fixation,
usually by a chemical or mixture of chemicals, permanently preserves the
tissue structure for subsequent treatments. This is a method of preserving the
protoplasmic structure and chemical composition of the tissue by interrupting
the dynamic process of the cell as quickly as possible and to stabilize and
harden the tissue preventing autolysis and postmortem decomposition.
Specimens should be immersed in fixative immediately after they are removed
from the body.
There are two types of fixatives:

1) Chemical fixation
Chemical fixation is a process of denaturing proteins and cross linking them to
provide a solid structure in place of the delicate membranes and interacting
molecules of the living cell. It is achieved with rapid penetration of living tissue
with fixative by immersion or perfusion. for immersion, small pieces of tissue
may be fixed by simply immersing them in fixative. In perfusion, tissues and

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organs can be fixed before they are removed from the body by pumping fixative
through the blood vessels that served them.
Perfusion is useful for fixing specimens too large to allow rapid fixative
penetration bt immersion (eg. in embalming). The commonly used fixatives are
Bouins, Formaldehyde and Glutaraldehyde. In practice, mixtures of fixatives
are often used.

2) Freezing (Physical Fixation)

Physical fixation involves freezing tissue to preserve its structure for either light
microscopy or Electron Microscopy. Rapid freezing at low temperatures in
liquid nitrogen helps formation of large ice crystals and related artefacts.
Freeing allows the tissue to be sectioned (or fractured) on freezing microtome
without chemical fixation, dehydration, or clearing. Sections are transferred
directly to the solutions for histochemical work. This is a rapid staining method
for diagnostic purposes

Fixation is used to:

1) Terminate cell metabolism,
2) Prevent enzymatic degradation of cells and tissues by autolysis (self-
digestion),
3) kill pathogenic microorganisms such as bacteria, fungi,and viruses, and
4) Harden the tissue as a result of either cross-linking or denaturing
protein molecules.

Dehydration
The purpose of this is to prepare chemically fixed tissue for infiltration with an
embedding medium by replacing water in the tissue with an organic solvent.
Ethyl alcohol is the most commonly used dehydrating agent. Others are like
acetone. Fixed tissue is immersed in a series of alcohol-water mixtures with an
increasing alcohol concentration (70% up to 100%)

Clearing
Clearing is done to replace the dehydrating agent with paraffin solvent that is
miscible both with the dehydrating agent and with the clearing – embedding
medium. Clearing renders the tissue transparent and the agents used include
Cedarwood oil, Benzene, Chloroform and Xylol
Infiltration
Permeating the tissue with a support media. Purpose: Replacement of clearing
agent with paraffin. Tissues are passed through a graded series of clearing
agent paraffin mixture. After completion, tissue is transferred to a clearing

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agent saturated with paraffin and then given two changes of paraffin. When
tissue is thoroughly infiltrated with paraffin, it is ready for embedding.

Embedding
It is to make the tissue firm by paraffin penetration of all intercellular spaces
and even into cells and prevent crushing or other tissue disruptions during
sectioning. This permits thin uniform sectioning.
In this process the infiltrated tissue is transferred to a container containing
molten paraffin which in turn is rapidly cooled and results in the formation of a
paraffin block of the tissue.

Sectioning
This cutting thin slices of tissue to permit microscopic analysis of their internal
structure. This allows for light or electrons to penetrate the specimen and form
an image.
A microtome is used to cut thin and uniform sections from the tissue. A
rotary microtome is ideal for cutting the tissues embedded in paraffin. The
cut sections are floated in warm water bath at 25-370C to spread the tissue
and avoid folds and mounted onto a glass slide using albumin to stick onto
microscope slide and later stained.

Staining
Staining is a method of dyeing the various cell organelles or its components by
using dyes or stain. Staining helps in the easy identification and study of the
cytological structures for microscopical studies. The choice of stain depends on
the cell component under investigation,the fixation used and its reaction with
the stain. Haematoxylin and eosin is routinely used for all teaching slides.
Morphological identification becomes easier. Haematoxylin is a basic dye and
stains the nucleus blue while eosin is an acidic dye and stains the cytoplasm
pink. Hematoxylin and eosin are used in histology primarily to display
structural features.
Tissue components that stain more readily with basic dyes are termed as
basophilic and those with an affinity for acid dyes are termed as acidophilic.
Examples of basic dyes are toludine blue and methylene blue.

Hematoxylin produces a very sharp blue to black chromatin stain when

oxidized to haematiein and combined with mordant. It colors basophilic

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materials in the tissue purple and blue, and is one of the simplest methods of
dyeing chromatin. Eosin colours cytoplasmic components pink or red.

TISSUES: CONCEPT AND CLASSIFICATION

Tissues are aggregates or groups of cells organized to perform one or more
specific functions. Tissues are classified into four different primary tissues of
the body having distinct structure and function.
Epithelium;
It is a layer or layers of cells which cover the body surfaces and line the body
tubes and membranes. They also form glands.
Connective tissues
It has supportive and connective function
Muscular tissues
It is specialized for producing movements because it possesses the property of
contraction.
Nervous tissue;
It is specialized for conduction, integration and relay of impulses.
Usually the structure and the composition of the extracellular matrix (the
noncellular substances surrounding cells) are the characteristics used to
classify tissue types. Epithelial and connective tissues are the most diverse of
the four tissue types and are component of every organ. Muscular and nervous
tissues on the other hand, are defined mainly according to their functions.

EPITHELIUM
It is derived from two Greek words, epi = upon and thelia meaning nipple. It is
a cellular layer which lines the body surface, skin and mucous membranes.
Cells may be arranged in a single layer or in multiple layers. The cells rest on a
basement membrane which is made of non-cellular amorphous substances
mainly mucopolysaccharides. The basement membrane is a specialized type of
extracellular material that is secreted primarily by the epithelial cells on the
side opposite their free surface and helps attach the epithelial cells to the
underlying tissues. Blood vessels do not penetrate the basement membrane to
reach the epithelium, thus all gases and nutrients carried in the blood must
reach the epithelium by diffusing across the basement membrane from blood
vessels in the underlying connective tissues. The most metabolically active cells
are close to the basement membrane. In stratified epithelia, cells die as they
move farther away from the basement membrane. The epithelial cells also form
glands by invagination (exocrine) or detachment (endothelium) which is
epithelial derivatives where the cells secrete specific substances. Epithelium in

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case of serous membrane is called mesothelium which in the case of those
lining the blood vessels, is referred to as endothelium.

Classification of epithelium
Epithelia are classified primarily according to the number of cell layers and the
shape of the cells.
1. On the basis of arrangement of cell layers
a) Simple epithelium- which consist of a single layer of cells, with each cell
extending from the basement membrane to the free surface.
b) Stratified epithelium – which consists, of more than one layer of cells and
only one of the layers is adjacent to the basement membrane.
c) Pseudo stratified epithelium- this consists of a combination of cells, with
all the cells attached to the basement membrane but only some of them
reaching the free surface. This epithelium is called pseudo stratified because
although it consists of a single layer, it appears multilayered as the height of
cells varies and also the arrangement of nuclei gives a stratified appearance.

2. On the basis of shape of cells

Categories of epithelium based on cell shape include;
a) Squamous – in which the height of cells is negligible as compared to width.
b) Cuboidal – height and width of cells are equal.
c) Columnar – the height of cell is much greater than width.

In most cases an epithelium is given two names with the first name indicating
the number of layers and the second indicating the shape of the cells. Using
this criterion, epithelia can be classified into the following types;
i) Simple squamous epithelium
Cells are flat, squamous or plate like. Nucleus is centrally placed and bulges

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the cell wall at free side. Thus, it gives the appearance of thelia or nipple. This
fact is responsible for the nomenclature of epithelium. Examples are, lining
epithelium of pleural, pericardial and peritoneal membranes where it is called
mesothelium or pavement epithelium, the inner lining of heart, blood vessels
and lymphatic where it is called endothelium. Simple squamous epithelium is
also found in some portions of nephron and labyrinth of internal ear.
ii) Simple cuboidal epithelium
Cells are arranged like cubical blocks on a basement membrane. Nucleus is
centrally placed e.g. thyroid gland and proximal renal tubule.
iii) Simple columnar epithelium
It is the chief secretory tissue of the body. It consists of a single layer of tall
prismatic cells resting on a basement membrane. The nuclei are basal i.e. near
the basement membrane. Simple columnar epithelium are found in most of the
parts of gut and genital system e.g. stomach, intestine, their glands, male
urethra, vas deferens, prostatic ducts, bulbo-urethral and greater vestibular
glands.
Stratified Epithelium
Stratified squamous epithelium
It consists of several layers of cell. The deepest layer consists of columnar cells
vertically arranged on a basement membrane. The middle layer or layers
contain polyhedral cells and superficial layers have flattened cells. The cells of
deep layer proliferate and undergo progressive change in morphology as they
pass towards the surface, where they are lost due to friction. This tissue is
specialized for withstanding friction. It is of two types;
a) Keratinized as in epidermis where the superficial squamous cells are
hardened due to a protein – keratin.
b) Non-keratinized e.g. cornea, esophagus, anal canal and vagina.
Stratified columnar and cuboidal epithelium
It consists of superimposed fusiform cells as in the membranous urethra.
Stratified cuboidal is found in the ducts of salivary glands and in pancreatic
duct.
Pseudo stratified epithelium
It has a single layer of cells set on a basement membrane but the height of the
cells varies. All cells do not reach the surface. The nuclei too are present at
different levels and therefore multiple rows of nuclei give a false appearance of
stratification. Examples are the lining epithelium of nasal cavity and most of
the male sexual ducts.
Transitional epithelium
Its appearance varies with the state of distension or contraction of the wall. It
is characteristics of ureters, urinary bladder and part of urethra. In the relaxed

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condition it has 5 to 6 layers, are pear-shaped with their conical ends touching
the basement membrane. The superficial cells are domed shaped with basal
surface touching the rounded ends of pear shaped cells of the second layer.
In distended condition, the cells of superficial layers get more flattened and
those of the middle layers get sandwiched between deeper cells.

Correlation of cell layer and cell shapes with function;

Simple epithelium with its single layer of cells is found in organs in which the
principal functions are diffusion (lungs), filtration (kidney), secretion
(glands), or absorption (intestines). The selective movement of materials
through epithelium would be hindered by a stratified epithelium, which is
found in areas where protection is a major function. The multiple layers of
cells in stratified epithelium are well adapted for a protective role because as
the outer cells are damaged, they are replaced by cells from deeper layers and a

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continuous barrier of epithelial cells is maintained in the tissue. Stratified
squamous epithelium is found in areas of the body where abrasion can occur
such as the skin, mouth, esophagus, anus and vagina.
Differential function is also reflected in cell shape. Cells involved in diffusion
and filtration are normally flat and thin. For example simple squamous
epithelium forms blood vessel and lymph capillaries, the alveoli (air sacs) of
the lungs, and parts of the kidney tubules.
Cells with the major function of secretion or absorption are usually cuboidal or
columnar. Their greater cytoplasmic volume as compared to that of squamous
epithelium is as a result of the presence of organelles responsible for the
tissue’s function e.g. pseudo stratified columnar epithelium, which secretes
large amount of mucus lines the respiratory tract and contains large mucus-
filled goblet cells which are specialized columnar epithelial cells.
Cell Surface
The surface of epithelial cells can be divided into three categories.
i) A free surface that faces away from underlying tissue
ii) A free that faces other cells
iii) A face that faces the basement membrane, the basilar surface.
Types of free surfaces include smooth, microvillar and ciliated. Smooth surface
reduces friction. Simple squamous epithelium with a smooth surface forms the
covering of serous membranes. The lining of blood vessels is a simple
squamous epithelium that reduces friction as blood flows through the vessels.
Microvilli greatly increase surface area and are found in cells involved in
absorption or secretion e.g. the lining of the small intestine. The free surface of
cells is covered with processes or projections from cell membrane, called cilia.
Cells are usually columnar in shape e.g. mucous membrane of lower part of
pharynx and of respiratory tract, tympanic cavity, auditory tube, uterine tube,
epididymis, ventricles of brain and central canal of spinal cord. Cilia propel
substances across the surface of the cell. Simple ciliated cuboidal, simple
ciliated columnar, and pseudo stratified ciliated columnar epithelia are in the
respiratory tract where mucus that contains foreign substances e.g. dust
particles is removed from the respiratory passages by the ciliary movements of
these tissues.

Functional Correlation of Epithelia

Epithelial tissues perform many functions and these include
Selective diffusion- Epithelium permits diffusion across it. The process of
diffusion is either facilitated or obstructed according to the requirement. Thus
epithelium acts as a selective barrier for materials diffusing across the
epithelial cells.

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Protection- It provide protection against mechanical trauma e.g. epidermis,
mucosa of mouth, upper esophagus, vagina and anal canal. These are
keratinized to withstand more trauma and also to provide protection against
drying, irritants and infection (stratified epithelium)
Transport- Mucus and particulate material is transported along the epithelial
surface e.g. respiratory and genital tract. This is mainly accomplished by
ciliated epithelium.
Excretion- Urine, sweat and carbon dioxide are diffused across the epithelial
surfaces. They filter the waste product of metabolism from blood.
Secretion- In glandular epithelium.
Absorption- As in intestinal epithelium
Sensory reception- Some epithelial cells are specialized for impulse
transmission e.g. taste buds, olfactory epithelium, nasal epithelium and the
organ of Corti in internal ear.
Lubrication- Mucus secreted by epithelial cells acts as lubricant.
Transitional epithelium has two important functions. It is capable of
distension and provides a waterproof surface impermeable to urine.

GLANDS
Glands are secretory organs. Most glands are composed primarily of epithelium
with a supporting network of connective tissues. The epithelial cells
constituting glands are capable of elaborating secretions. Those glands in
which the secretion emerges on to epithelial surface through ducts are known
as exocrine. Those from which the secretion passes directly to bloodstream
and carried throughout the body are termed as endocrine glands. All glands
are essentially epithelial, formed by invagination (folding in) of the surface
epithelium. Exocrine glands maintain contact with the surface by means of
ducts and discharge their secretions on that surface while the endocrine glands
are completely separated from the surface lining as they lose their ducts and
are therefore called ductless glands.

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Classification of Glands

According to the mechanisms of secretion

1. Exocrine glands
Elaboration of secretions in exocrine glands is by three different mechanisms.
In general, the secretory materials formed in the cells are extruded by reverse
pinocytosis.
a) Merocrine
Cells remain intact while secretory granules are released. Merocrine glands
such as water producing sweat glands and the exocrine portion of the pancreas
secrete products with no loss of actual cellular material.
b) Apocrine
The basal portion of the cells with nucleus remains intact while the luminal
portion with the secretory granules is extruded with the cells in the secretion.
c) Holocrine
Whole cell is discharged together with the secretion e.g. sebaceous glands of
the skin shed entire cells. Substances accumulate in the cytoplasm of each
epithelial cell, the cell ruptures and dies, and the entire cell becomes part of

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the secretion.

ii) Shape of glands

a). Unicellular- some exocrine glands can be composed of a single cell e.g. the
goblet cells of the gastro-intestinal mucus membrane or respiratory system
b). Multicellular – The glands is composed of many cells which co-operate in
making a complex gland. Multicellular glands can be classified further
according to the structure of their ducts. Glands that ducts with few branches
are called simple, and glands with ducts that branch repeatedly are called
compound. Further classification is based on whether the duct ends in tubules
(small tubes) or sac-like structures called acini or alveoli.
They are therefore classified as;
i. Tubular; - secretory cells are arranged in an elongated tube with a central
lumen. It may be straight, coiled or branched.
ii. Acinar; - the cells are arranged in rounded sacs.
iii. Alveolar – cells are arranged in flask shaped sacs’

Simple exocrine glands include-:

a). Tubular – in which a single straight tubules secretes on the surface e.g.
crypts of Lieberkuhn.

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b). Coiled tubular- in which a spiral tubular gland secretes by a single duct e.g.
sweat glands.
c). Branched tubular-in which ramifying tubular branches secretes by short
ducts e.g. glands of the body stomach.
d). Acinar- in which a single acinus secretes on a surface e.g. urethral glands.
e). Branched acinar- in which multiple crescentic acini secrete into a straight
duct e.g. sebaceous glands.
Compound exocrine glands include:
a). Compound tubular-straight or coiled tubular terminal secretory portions
join the main duct e.g. glands of Brunner of duodenum.
b). Compound Acinar- acini lie at the ends of branches of the main duct e.g
exocrine pancrease.
c). Compound Tubular-alveolar- in which both tubular and alveolar glands lie
at the ends of branched tubules e.g. parotid gland.

iv) Nature of secretion

a). Mucus – these produce a viscous, slimy and chemically inert mucus e.g.
palatine glands, glands of cervix, uteri and goblet cells.
b). Serous – these produce a thin watery albuminous secretion which usually
contains enzymes e.g. parotid gland and pancreas.
c). Sero-Mucus- the secretion is mixed owing to the presence of both serous
and mucous types of cells e.g. sublingual and submandibular glands.
d). Cytogenic – the gonads (testis and ovary) produce very highly specialized
secretions which consists of living cells (sperm and ova). They are therefore
called cytogenic glands.

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2. Endocrine glands
Endocrine secretions in contrast, pass from gland cells to vessels by simple
molecular diffusion, by pinocytosis or by trans-membrane transport.

3. Paracrine glands
This is a new addition to the classification of glands referred to some hormone
secreting cells present amongst the epithelial cells of gut and lung alveoli. Their
secretions pass on to their neighboring or adjacent cells by simple diffusion.
They cannot be called endocrine cells because their secretions are poured into
blood vessels though in practice they are taken as endocrine cells and are
included in endocrine system because of their hormone secreting function.

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