Introduction To Human Histology L

Introduction
to human histology
What is histology ?
• The study of microscopic (microanatomy) of
body structure.
• Observe condition of cells and tissue under
disease free condition.
Histopathology ?
• it is a branch of science which deals with the
study of disease in a tissue section.
Histochemistry ?
• means study of chemical nature of the tissue
components by histological methods.
• The cell is the single structural unit of all
tissues. The study of cell is called cytology.
• A tissue is a group of cells specialized and
differentiated to perform a specialized
function.
Types of Histological preparation
1. Whole mounts- These are preparation entire
animal eg. fungus, parasite. These preparations
should be no more than 0.2-0.5 mm in
thickness.
2. Sections- The majority of the preparations in
histology are sections. The tissue is cut in about
3-5 mm thick pieces processed and 5 microns
thick sections are cut on a microtome.
3. Smears- Smears are made from blood, bone
marrow or any fluid such as pleural or ascetic
fluid. These are immediately fixed in alcohol to
presence the cellular structures are then
stained.
Slide preparation :
• The microscopic examination of tissue sections and their
proper preparation is essential in the study of tissue
morphology.
• A basic knowledge of the various types of microscopes
and histological techniques is important to learn ,this
helps to study the structure and function of tissues .
• The most common types of microscopes for studying
tissues are:
- light microscope – not better than 6µm
- electron microscope –for subcellular structures
Histotechniques steps
1- Fixation.
2-Dehydration.
3-Clearing (dealcoholization).
4-Embedding.
5. Sectioning.
6. Staining.
FIXATION
• It is a complex series of chemical events which
brings about changes in the various chemical
constituents of cell like hardening, however the
cell morphology and structural detail is
preserved.
• Unless a tissue is fixed soon after the removal
from the body it will undergo degenerative
changes due to autolysis and putrefaction so that
the morphology of the individual cell will be lost.
• Principle of fixation- The fixative brings
about crosslinking of proteins which
produces denaturation or coagulation of
proteins so that the semifluid state is
converted into semisolid state; so that it
maintains everything in vivo in relation to
each other. Thus semisolid state facilitate
easy manipulation of tissue.
Aims and Effects of fixation
1. To preserve the tissue in as lf like manner as
possible.
2. To prevent postmortem changes like autolysis
and putrefaction.
3. Preservation of chemical compounds and
microanatomic constituents so that further
histochemistry is possible.
4. Hardening
5. Solidification: Converts the normal semifluid
consistency of cells (gel) to an irreversible
semisolid consistency (solid).
6. Effects of staining - certain fixatives like
formaldehyde intensifies the staining character
of tissue especially with haematoxylin.
• The fixative should be at least 15-20 times the

bulk of tissue.
Reagents employed as fixatives
• Formaldehyde - Formaldehyde is a gas but is
soluble in water to the extent of 37-40%.
• It preserves the proteins by forming cross
linkage with them and the tissue component.
• Formalin on prolonged exposure can cause
either dermatitis its vapour may damage the
nasal mucosa and cause sinusitis.
Time required for fixation.
• At room temperature - 12 hours
• For small biopsies - 4-6 hours
• At 65°C fixation occurs in - 2 hours
Alcohol (Ethyl Alcohol)
• Absolute alcohol alone has very little place in
routine fixation for histopathology.
• It is slow to penetrate, hardens and shrinks
the tissue.
• Ethanol preserves some proteins in relatively
undenatured state so that it can be used for
immunofluorescence or some histochemical
methods to detect certain enzymes.
Histological
processingcassettes
Dehydration
• The tissue is transferred through a series of
increasingly concentrated alcohol solutions,
ending in 100%, which removes all water.
• Ethanol, is commonly used for the removal of
fixative agent and water from the tissue .
• Gradually increasing percentages of ethanol
(60% ,70% , 80%,95% and absolute alcohol)
are used.
Clearing
• The ethanol is replaced by an organic solvent
miscible with both alcohol and the embedding
medium (paraffin) , a step referred to as
clearing because infiltration with the reagents
used , gives the tissue a translucent
appearance.
• Replacement of alcohol in tissue by clearing
fluid like xylene
Embedding
• The paraffin-infiltrated tissue is placed in a
small mold with melted paraffin and allowed
to harden.
• when solidified, it will provide support
during sectioning.
Sections
• The hardened block with tissue and
surrounding embedding medium is trimmed
and placed for sectioning in an instrument
called a microtome .
• Paraffin sections are typically cut at 3-10 μm
thickness (thin strips) for light microscopy .
Mounting
• The sections are placed (mounted) on a glass
slides coated with suitable adhesive .
• The most commonly adhesives used :
1. Albumin
2. Gelatin
3. Starch
4. Cellulose
Staining
• The sections, as they are prepared, are
colorless and different components cannot be
appreciated. Staining them by different colure
dyes, having affinities of specific components
of tissues, makes identification and study of
their morphology possible.
• Most common dye used are hematoxylin and
eosin.
• Hematoxylin is a base , it colors the acidic
components of the cells by bluish color , so
the nucleus stains blue .
• Eosin is an acid ,it dyes the basic components
of the cells by pinkish color , so cytoplasm
stains pink.
Other stains
• Giemsa stain for bone marrow and blood
smear.
• Trichrome stain: distinguish between collagen
fibers (green or blue) from muscle fibers(red).
• Silver stain: used to demonstrate proteins,
such as type III collagen and reticulin fibers ,
by deposition of metallic silver particles on
the targets of interest ,(black deposit).
Silver stain
Giemsa stain
• Stained slide covered with cover slip.
• Mounting media used mostly DPX or Canada
balsam
DECALCIFICATION
• Decalcification is a process of complete removal
of calcium salt from the tissues like bone and
teeth and other calcified tissues following
fixation.
• Decalcification is done to assure that the
specimen is soft enough to allow cutting with the
microtome knife. Unless the tissues in completely
decalcified the sections will be torn and ragged
and may damage the cutting edge of microtome
knife.
• The Criteria of a good decalcifying agents
area.
1. Complete removal of calcium.
2. Absence of damage to tissue cells or fibers.
3. Subsequent staining not altered.
4. Short time required for decalcification.
• various agents used for decalcifying are :
1- Nitric acid
2- Hydrochloric acid

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• The fixative should be at least 15-20 times the

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