Histology

Histology: is the scientific study of microscopic structures of tissues and organs ofthe body
.also called microscopic anatomy (histos = tissue; logia = science or study)

In human's body, there are about 200 different types of cells and consist of around 9 billio
.Cells

Cell: is the basic structural, functional, and biological unit of all kmown organisms. A cell is
the smallest unit of life. Cells group together to form tissues, which in turn group together
to form organs, such as the heart and brain
.
Tissue: is a group of cells that have similar structure and that function together as a unit
There are four main tissue types in the body: epithelial, connective, muscle, and nervous
Each is designed for specific functions
.
Tissues that can be taken to exam for histology and histopathology are Biopsy
and Autopsy.

Biopsy: is a sample of tissue from a living organism which is used as a

diagnostic tool. The aim of biopsy is to identify various diseases. It is a
diagnostic method where a very small piece of tissue is taken from various
parts of body depending upon the disease (it could from skin, mouth, bone or
any of the internal organs

 
 Microscope:
A microscope is a laboratory instrument used to examine objects that are too
small (cells and tissues) to be seen by the naked eye. Microscopy is the science of
investigating small object and structures using a microscopic
.
1. Eyepiece or Ocular Lens
Eyepiece lens magnifies the image of the specimen. This part is also known as
ocular. Most school microscopes have an eyepiece with 10X magnification.
Eyepiece Tube or Body Tube The tube holds the eyepiece.
2. Nosepiece
Nosepiece holds the objective lenses and is sometimes called a revolving turret. You
choose the objective lens by rotating to the specific lens one you want to use
.

3. Objective Lenses
Most compound microscopes come with three or four objective lenses that revolve en
the
nosepiece. The most common objective lenses have power of 4X, 10X and 40X.
Combined
with the magnification of the eyepiece the resulting magnification is 40X, 100X and
400X
magnification. Total magnification is calculated by multiplying the power of the
eyepiece by the power of the objective lens.

 
4. Stage
The stage is where the specimen is placed. This place is for observation
.
5. Stage Clips
Stage clips are the supports that hold the slides in place on the stage.
 
6. Diaphragm (sometimes called the Iris) The diaphragm controls the amount of
light passing through the slide. It is located below the
stage and is usually controlled by a round dial. How to set the diaphragm is
determined by the magnification, transparency of the specimen and the degree of
contrast you wish to hav in your image. Also called the condenser diaphragm
.

 
7. Illuminator
Most light microscopes use a low voltage bulb which supplies light through the
stage ando nto to the specimen. Mirrors are sometimes used instead of a built-in
light. If your microscope has a mirror, it provides light reflected from ambient light
sources like classroom lights or sunlight if outdoors
8. Coarse focus
Coarse focus moves the stage to provide general focus on the specimen. When
bringing a specimen into focus, the course dial is the first one used.

 
9. Fine focus
Fine focus moves the stage in smaller increments to provide a clear view of the
specimen When bringing a specimen into focus, the fine focus dial is the second
one used
.
 
10. The base is the main support of the microscope. The bottom, where all the
other parts the microscope stand.
Electron microscope (EM): is a technique for obtaining high resolution images of
biological and non-biological specimens. It is used in biomedical research to
investigate the detailed structure of tissues, cells, organelles and macromolecular
complexes
Histopathology processes
Microscopic analysis of cells and tissues requires the preparation of very thin, high quality
sections (slices) mounted on glass slides and appropriately stained to demonstrate
normal and abnormal structures.

 
Paraffin Tissue Processing:
1- Fixation and Decalcification
2- Dehydration (Tissue Processing)
3- Clearing
4- Embedding (Blocking)
5- Cutting Sections (Microtome)
6- Staining
7-Mounting.
 • Fixation and Decalcification

The most common fixative for light microscopy and Histology is 10% neutral
buffered formalin (10% NBF). To prevent post-mortem changes like autolysis and
putrefaction
.
Decalcification is a process of complete removal of calcium salt. Nitric acid- 5-
10% from the tissues like bone and teeth and other calcified tissues following
done to assure that cutting with the microtome knife.
Dehydration (Tissue Processing)
The aim of Tissue Processing is to remove water from tissues and replace with
a medium
that solidifies to allow thin sections to be cut. The most common dehydrant is
Ethanol
Note: Tissues are dehydrated through four to seven changes of dehydrants and
the last
should always be fresh. 50%-70% ethanol, progressing through 90%-95%/
ethanol, then two or three changes of absolute ethanol before proceeding to
the clearing stage
Clearing (Removal of Alcohol)
Clearing is the transition step between dehydration and infiltration with the
embedding medium. It should be miscible with both solutions (dehydrant and
the embedding medium), The most common Clearing agent is Xylene.
Infiltration and Embedding (Blocking)
After the tissues have been dehydrated and cleared must be supported in a
hard matrix to allow sufficiently thin sections to be cut. For light
microscopy, paraffin wax is most frequently used. (Parablast). Infiltrating
and embedding medium should be fluid when hot and solid when cold.

• 
Embedding (Blocking):
As soon as the tissue is thoroughly infiltrated, it is ready to be embedded. The
tissue is placed in a small container on paper box-already filled with melted
embedding material and whole is cooled rapidly.
Cutting Sections (Microtome)
Routine Paraffin Embedded tissues are normally cut between 3-5 um (microns)
(1000microns =1000 micrometres =1 mm) thick for light microscopy.
for electron microscopy 80-100nm (nanometre) (1,000,000 nanometres = 1mm
6 Staining
Stains are used as an aid and a diagnostic tool for a final diagnosis. The
staining process will allow us to physically be able to visualize a paraffin
section slide under the microscope
.
The two stains most widely used for routine work are hematoxylin and cosin
(commonly abbreviated as H & E). Hematoxylin stains negatively charged
structures, such as DNA, a blue color. Eosin imparts a pink color to most of the
other cell components

.
 
7 -Mounting
The final step in this procedure is to permanently mount the sections
under a coverslip. The ideal mounting medium should not distort the
stain colour or yellow and become brittle with age
.
 
Permanent mounting media:
1- Distrene-Plasticizer-Zylene (D.P.X) is a synthetic media.
2-Canada balsam is a natural media.
Freezing section:
The frozen section is the rapid tissue section by cooling the tissue with the help
of cryostat toprovide immediate report of the tissue sample. The cryostat is the
instrument to freeze the tissue and also to cut the frozen tissue for microscopic
section.
 
Freezing microtome: This microtome is used for cutting thin sections of fresh,
frozen tissue.
 
Freezing section:
The frozen section is the rapid tissue section by cooling the tissue with the help
of cryostat toprovide immediate report of the tissue sample. The cryostat is
the instrument to freeze the tissue and also to cut the frozen tissue for
microscopic section.
 
Freezing microtome: This microtome is used for cutting thin sections of fresh,
frozen tissue.
 
Stains (dyes)  
Classification of dyes
1- Natural dyes (Hematoxylin, Cochineal, Carmine, Berberine and Orcein
stain)
2- Synthetic dyes
a- Acidic dyes (Eosin, Aniline Blue, Alizarin red).
b- Basic dyes (Hematoxylin, Safronin, Methyl green).
c- Neutral dyes (Giemsa, Leishman, Sudan Blue and Black).
Stains and their uses
1- Hematoxylin and Eosin (H&E stain). Hematoxylin, a basic dye, stains nuclei blue;
eosim,
an acidic dye, stains the cytoplasm pink. This is a General Morphology stain.
2-Papanicolus stain used for Cytology.
3-Giemsa stain used for cytology blood cells.
4-Massons Trichrome stain used for Connective tissue.
5-Gomori stain used for Reticulum.
6- Holezinger and Oil red stam used for Lipids and Fatty acids.
7- Methyl Green Pyronin used for DNA and RNA.
8- Jones Hexamine silver used for Basement Membranes.
9- Hematoxylin and Van Gieson used for Collagen.
10- Thionin stain used for Bone tissue.