HISTOLOGY AND ITS METHODS

MARK CHRISTIAN C. AMABILIS, RMT

CLINICAL INSTRUCTOR
COMMUNITY EXTENSION FOCAL PERSON
 Preparation of Tissues

The most common procedure used in histologic

research is the preparation of tissue sections or
slices that can be studied with the light microscope.
Under the light microscope, tissues areexamined
visually in a beam of transmitted light. Because most
tissues and organs are too thick for light to pass
through them, they must be sliced to obtain thin,
translucent sections that are attached to glass slides
for microscopic examination.
 Preparation of Tissues

• The ideal microscopic preparation is preserved so that the

tissue on the slide has thesame structure and molecular
composition as it had in the body. However, as a practical
matter, this is seldom feasible, and artifacts, distortions,
and loss of components due to the preparationprocess
are often present.
• Ø The objective of a histology is to lead the student to
understand the microanatomy of cells, tissues, and
organs and to correlate structure with function. The
methods used by histologists areextremely diverse.
• These auxiliary techniques include:
o• Light and Electron Microscopy
o• Autoradiography
o• Enzyme Histochemistry
o• Cell and Tissue Culture
o• Visualization of Specific Molecules
onterpretation of Structures and Tissue Sections
 • Autoradiography

• is a method of localizing newly synthesized

macromolecules in cells or tissue sections
• It makes use of a photographic emulsion placed over a
tissue section to localize radioactive material within
tissues
 HISTOPATHOLOGIC TECHNIQUES:

• 1. Fixation
• 2. Dehydration
• 3. Clearing
• 4. Infiltration
• 5. Embedding
• 6. Trimming
• 7. Section-Cutting
• 8. Staining
• 9. Mounting
• 10. Labeling
 1. Fixation

• 1. Fixation
• Small pieces of tissues are immersed in a
solution called fixative after removal from the
body. ·
• To avoid tissue digestion by enzymes present
within the cells or bacteria
• To preserve cell and tissue structure
• 2. Dehydration
• Tissue is transferred through a series of
increasingly concentrated alcohol solutions
called as dehydrating agents, ending in 100%,
which removes all water.
• 3. Clearing
• Alcohol is removed in the tissue by immersing
in a clearing agent.
• 4. Infiltration
• Tissue is then placed in melted paraffin until it
becomes completely infiltrated with the substance.
• 5. Embedding
• The paraffin- infiltrated tissue is placed in a mold
with melted paraffin and allowed to harden
• Note: The medium used in infiltration of tissue is
the same medium used for embedding.
• 6. Trimming
• The resulting paraffin block is trimmed to expose
the tissue for sectioning (slicing) on a microtome.
• 7. Section-Cutting
• Tissue block is sliced into thin films using a
microtome which are placed on glass slides and
• allowed to adhere, deparaffinized, and stained.
• 8. Staining
oMethods of staining have been devised that not only
make the various components conspicuous but also
permit distinctions to be made between them.
oCell components with a net negative charge (anionic)
stain more readily with basic dyes and aretermed
basophilic
oCationic components have affinity for acidic dyes and are
termed as acidophilic
• 9. Mounting
• Stained tissue slides are mounted with a
cover slip using a mounting media.
• 10. Labeling
• Tissue slides are labeled on the frosted areas
with assigned tissue numbers or codes.
Tissue
• slides should be properly labeled
 FIXATION

• FIXATION
• ✔ Preserve tissues structure and prevent degradation by
enzymes released from the cells or microorganisms by using
stabilizing or cross-linking compounds called: Fixatives
• ✔ Primary reason: Preserve the morphologic and integrity of the
cells in as life-like manner as possible
• ✔ Secondary reason: Harden and protect tissue from trauma of
f urt her handling, so t hat it is easier t o cut during gro s s
examination
• ✔ Most common fixative: 10% Formaldehyde
• DECALCIFICATION
• ✔ Process of complete removal of calcium or lime salts from tissues ( Bones,
Teeth, Calcified tissues) after fixation
• ✔ Should be done after Fixation and before Impregnation to ensure and
facilitate normal
cutting of sections
• ✔ Most Common Decalcifying agent: 5-10% Nitric Acid
• DEHYDRATION
• ✔ Process of removing water from the tissue following fixation in preparation
for wax impregnation
• ✔ Done by placing fixed tissues in Ascending grades of Dehydrating Fluid
(75% - 95% - 100%)
• ✔ Most Common Dehydrating agent: Ethanol
• CLEARING/DEALCOHOLIZATION (transparent)
• ✔ Process wherein alcohol/ dehydrating fluid is removed
from the tissue and replaced with an intermediate solvent
that is fully miscible with both ethanol and paraffin wax.
• ✔ Most Common Clearing Agent: Xylene
• INFILTRATION/IMPREGNATION
• Process whereby the clearing agent is completely
removed from the tissue and replaced by a medium that
will completely fill all the tissue cavities.
• ✔ Most Common Infiltrating agent: Paraffin Wax
• EMBEDDING/CASTING
• ✔ Paraffin impregnated tissue are placed into a
mold containing the embedding medium together
with their proper labels and immersed in a melted
paraffin at a temperature bet 5- 10C above the MP
of the paraffin wax
• ✔ Most Common Embedding Medium: Paraffin
Wax
• TRIMMING
• ✔ Process of removing excess wax after
embedding
• ✔ Tissue should be surrounded by at least 2 mm of
wax
• SECTIONING/CUTTING
• ✔ Process wherein a processed tissue is cut into
uniformly thin sections using MICROTOME to
• facilitate studies under the microscope
• STAINING
• ✔ Process of giving color to the sections to see and study
the architectural pattern of the tissue
• and physical characteristics of the cells
• ✔ Hematoxylin (Nucleus) and Eosin Stain (Cytoplasm)
• ✔ Periodic Acid Schiff = Glycogen
• ✔ Sudan Black = Lipids
• ✔ Fuelgen =DNA
• MOUNTING
• ✔ Process of putting cover slip on the stained tissue using mounting medium to
stick the cover slip on the slide. It is done to facilitate handling and to prevent
damage to the section
• ✔ Mounting Medium: Aqueous Mounting Medium and Resinous Mounting
Medium
• RINGING
• ✔ Process of sealing the margins of the cover slip to prevent escape of
fluid/semi fluid mounts
• and evaporation of the mountant.
• ✔ It is done to immobilize the cover slip and to avoid sticking of the slides upon
storage
• LABELING
• ✔ Process of indicating a year and specimen number on
end of the prepared slide for proper identification
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