Dr Mohamed Serar Yusuf
Msc, Bsc, Ascpi ,UMST/Sudan
Welcome to the classes of Histology.. We′ll talk
about histology.
What is histology?
How to study it ?
Key points
& Definition and study contents of histology.
& Paraffin section and HE staining.
& Light and electron microscopy.
& Histochemistry and immunohistochemistry.
& Observation of living tissue
Histology & Its Study
Histology is a branch of Anatomy.
Anatomy:
Gross anatomy
Microscopic anatomy
Histology is a science which study the
microstructures and their functions of human being.
How can we observe microstructures?
We need a microscope to observe microstructures.
We study microstructures at three structural
levels: cells, tissues and organs.
Cells
The cell is the smallest structure and functional
unit of living organisms. There are many kinds of
cells in human body such as epithelial cells,
fibroblasts, plasma cells and nervous cells .
cell.ppt
Tissues
Groups of cells and intercellular substances make up
the body tissues. There are four main types of tissue:
epithelium, connective tissue, muscle tissue and
nervous tissue. connective tissue.ppt
Organs
Several different kinds of tissue are further organized
to form organs ( such as heart, stomach or liver) .
Thus the human body may be examined at three
structural levels: cells, tissues and organs.
System
Several organs with related functions together form
systems. For example, the kidneys, ureters, urinary
bladder and urethra constitute the urinary system.
Why need we study histology?
Histology is the study of normal structure and
function of cells, tissues and organs in the body.
Learning normal tissue architecture and
understanding its relation to normal tissue and organ
function will provide the student with the foundation
for recognizing the hallmarks of pathology and
disease.
How to study histology?
Histology studies the microstructures. So, we should
have the aid of microscope to study. Several types of
microscopes are available. According to the light
source used, microscopes can be basally classified as:
Light microscope(abbreviate LM)
Electron microscope(abbreviate EM)
Light microscope
With the LM, stained specimens are usually examined
by means of light that passes through the specimens,
and the magnification of 1 000 times can be achieved.
The maximal resolving power of LM is approximately
0.2um. That means that the smallest distances
between two particles at which they can be seen as
separate objects is 0.2um.
Um: micron
The EM uses an electron beam instead of light; and
electromagnetic fields in place of lenses. With the
EM magnification of 100 000(hundred thousand)
times can be achieved. The maximal resolving power
is 0.2 nm under the EM.
The structures of a cell or tissue as seen with the EM
are referred to as ultrastructures.
There are two kinds of EM:
The transmission electron microscope (abbreviate
TEM) and the scanning electron microscope (SEM).
The three dimensional images of a structure can be
obtained using SEM.
The figures of TEM & SEM.ppt
Traditional Histological Methods
Preparation of a specimens for LM
Before a piece of tissue can be observed with a light
microscope, it must be made into a section. The
routine histological preparation for light microscope
examination is a paraffin sections stained with
haematoxylin and eosin (HE staining).
The procedure for making a section is as follows:
Tissue collecting —Fixation —Dehydration
—Clearing —Embedding—Sectioning —Staining
Tissue collecting Fresh, small tissue blocks are get
from a organ. The size of a tissue block should be less
than 1.2cm×0.5cm×0.2cm. （ centimeter ）
Fixation Then put the tissue blocks into a fixative.
The process of fixation preserves a tissue by
denaturing its proteins. Numerous fixatives are
known, the most commonly used being 4%
formaldehyde (4% formaldehyde is also called
formalin).
Dehydration Then use ethyl alcohol to get rid of
water from the tissue blocks.
Clearing Then use xylene to get rid of alcohol.
*alcohol and xylene are embedding mediums
Embedding Before a tissue can be sectioned it has to
be given a firm consistency. One way of doing this is
embedding a tissue in paraffin wax .
The procedure of
embedding
Heat the paraffin firstly,
make it melt, then spill it
into a box.
Put tissue block into
melted paraffin, allow
paraffin harden, the
tissue block is embedded
in.
Sectioning Usually sections 3 ～ 8micron(μm) thick
are cut with a microtome and mounted on glass slides.
When the sections are dry, they are ready for staining.
Staining
Numerous staining methods are available for
demonstrating specific tissue elements. The most
extensively used staining method is HE Staining .
HE Staining That the sections are stained with
haematoxylin-eosin is called HE staining.
Haematoxylin is a basic dye. It can bind to the acidic
components of cells and tissues, which then show a
blue colour. Such components, e.g. nuclei, are said to
be basophilic. RER and ribosomes in cytoplasm are
basophilic too.
Eosin is an acidic dye. It can bind to basic
constituents and give them a pink colour. Such
constituents, e.g. cytoplasm and most of other
components, are termed acidophilic.
HE stain.ppt RER-HE.ppt
Some components can not be stained by
haematoxylin nor by eosin. Those components are
termed neutrophilic.
Except sections we can also get a specimen as
follows:
Smear preparation is used to observe blood or other
secretions.
Spread preparation is used to observe mesothelium
and connective tissue.
Grinding preparation is used to observe bone or
tooth.
Histochemical techniques
Histochemical techniques or cytochemical techniques
combine histological methods with chemical and
biochemical methods and reveal the chemical
composition of tissues and cells in situ. Many
substances, such as proteins, amino acids, nucleic
acid, lipids, carbohydrates and enzymes et al , can be
detected in situ by histochemical and cytochemical
techniques.
PAS reaction
The periodic acid-Schiffs reagent (PAS) reaction is
one of histochemical techniques most extensively
used to localize polysaccharides, such as glycogen
and glycoprotein, in tissues and cells.
Immunocytochemical technique
antigen-antibody reaction.
Specific molecules(antigen) within cells can be
identified by treating tissue sections with antibodies
specific to the molecules.
The technique enables chemical substances to be
localized in cells with great precision. Such
studies have greatly enhanced our knowledge of
chemical transformations taking place within
cells.
immunocytochemistry.ppt
In situ hybridization
In situ hybridization(abbreviate ISH), also termed in
situ hybridization histochemistry(ISHH), is a
method for detection of specific RNA or DNA
sequences directly in cells or tissue sections.
Observation of living cells and tissue
Vital staining
Dyes used for vital staining are usually neither toxic to
living cells nor destroyed by them. When the dyes are
injected into living animals certain cells or structures
can be stained by the dyes or can be visualized by their
selective absorption of colouring substance. For
example, trypan blue injected into living animals is
removed from the blood by macrophages and
accumulated in the their cytoplasm. This renders the
cytoplasm of macrophage conspicuous.
Vital staining of trypan blue. Macrophages engulf
trypan blue granules in their cytoplasm.
Cell and tissue culture
Cell and tissue culture is a technique for the study of
living cells. Isolated cells or fragments of tissues are
cultured in a sterilized culture medium at the
appropriate temperature. Since the medium
contains essential nutrients, such as amino acids,
vitamins, et cetera, cells and tissues grow in vitro.
This technique is quite useful in detecting the effect
of various reagents on living cells.
The photo of cultured epithelia cells
QUESTIONS
1. What is histology?
2. What is HE stain?
3. What are differences between the light and
electron microscopy?
4. What components can the PAS reaction detect?
5. How does histologist study the living cells and
tissues?
