HISTOLOGY

Gianina D. Dacuya, RMT

 Introduction to Histology
• Branch
of anatomy that deals with the
STUDY OF TISSUES
• Notonly tissues but also that of cells,
organs and system
 HISTOTECHNIQUE

A method of preparation of tissue for

microscopic examination
 STEPS FOR PARAFFIN
METHOD

1. Procurement of tissue specimens

STEPS FOR PARAFFIN
METHOD
2. FIXATION- submerging the
specimen in chemical substances in
order to PRESERVE THE TISSUE.
 STEPS FOR PARAFFIN
METHOD
3. DEHYDRATION – Immersing the tissue
in increasing concentration of alcohol to
REMOVE WATER from the tissue.
 STEPS FOR PARAFFIN
METHOD
4.CLEARING (DEALCOHOLIZATION) -
Since alcohol is insoluble with paraffin, it must
be replaced by a clearing agent so that it can be
IMPREGNATED WITH PARAFFIN WAX.
 STEPS FOR PARAFFIN
METHOD
5. INFILTRATION – Removal of clearing
agent. Its purpose is TO FILL THE
TISSUE GAP with a solid medium (wax)
 STEPS FOR PARAFFIN
METHOD
6. EMBEDDING – Placing the tissue in
paraffin blocks. Paraffin penetrates all
intercellular spaces and even into the cells
making the tissue MORE RESISTANT TO
SECTIONING.
 STEPS FOR PARAFFIN
METHOD
7. SECTIONING – CUTTING OF TISSUE by
use of MICROTOME about 3-5 um in
thickness. The cut tissue, paraffin ribbonettes
are placed in a basin of water.
 STEPS FOR PARAFFIN
METHOD
8. STAINING :
• Stainsthat differentiate acidic and
basic components of the cell
• Stains
that differentiate fibrous
component of the matrix
• Metallic
salts that precipitate tissue
forming metal deposits on them
STEPS FOR PARAFFIN
METHOD

Hematoxylin and Eosin Stain (H & E Staining)

(MOST COMMONLY USED

STAINING PROCEDURE)
 STEPS FOR PARAFFIN
METHOD
8. STAINING :
BASIC - Stains basophilic tissue components
• Basicdyes:
 Toluidine blue
 Alcian blue
 Methylene blue
ACIDIC - Stains acidophilic tissue components
• Acidic dyes
 Eosin
 Acid function
 Orange G
 STEPS FOR PARAFFIN
METHOD
8. STAINING :
• Orceins Weigert’s elastic stain
 Elastic stain
• Silver stain
 Reticular fibers
• Iron hematoxylin
 Striation of muscles, nuclei, erythrocytes
• Periodic Acid Schiff
 Glycogen, carbohydrate-rich molecules
• Wright and Giemsa
 Differential staining of blood cells
STEPS FOR PARAFFIN
METHOD
9. MOUNTING – Placing a cover slip
with a few drops of CANADA BALSAM
as a mounting medium on processed
tissue.
STEPS FOR PARAFFIN
METHOD
10. LABELLING – Label the organ on the prepared
slides.
Microscopy
ANATOMY OF MICROSCOPE
• EYEPIECE – Ocular
• BINOCULAR – Having two oculars or eyepieces
• COARSE ADJUSTMENT – Control that adjusts
position of microscope objectives; used to initially
bring objects into focus
• CONDENSER – Apparatus located below the
microscope stage that directs light into the objective
 ANATOMY OF MICROSCOPE
• FINE ADJUSTMENT – Control that adjusts position of
microscope objectives; used to sharpen focus
• IRIS DIAPHRAGM – Device that regulates the amount
of light that strikes the specimen being viewed through
the microscope.
• LENS – A curved transparent material that spreads or
focuses light
• MICROSCOPE ARM – The portion of the microscope
that connects the lenses to the base
• MICROSCOPE BASE – The portion of the microscope
that rests on the table and supports the instrument
 ANATOMY OF MICROSCOPE
• NOSEPIECE – Revolving unit to which microscope
objectives are attached
• OBJECTIVE – Magnifying lens that is closest to the
object being viewed with a microscope
• PARFOCAL – Having objectives that may be
interchanged without varying the focus of the
instrument
• RESOLVING POWER – The ability of a microscope
to produce a clear image, resolution
• STAGE – Platform that holds object to be viewed
microscopically
DIFFERENT
TYPES OF
MICROSCOPE
 Types of Microscope

1. COMPOUND MICROSCOPE

Also called a “light microscope” or “optical

microscope”. The principle is the refraction light .
Types of Microscope

2. ELECTRON MICROSCOPE
Function on the principle that a beam
of electron can be deflected by
electromagnetic fields in a manner
similar to light deflection in glass.
Types of Microscope

3. PHASE CONTRAST MICROSCOPE

It is useful for the study of unstained cells, either
living or fixed. Also, useful in the study of
mitosis that renders the chromosomes and
other cell organelles darker that the
surrounding cytoplasm
Types of Microscope

4. FLOURESCENT MICROSCOPE

Useful in localizing antigen-

antibody complexes within
the tissues.
 Types of Microscope

5. POLARIZING MICROSCOPE
Detects linearly oriented structures of living cells in
tissue cultures or in fixed stained preparations. Useful
in viewing the spindle fibers of dividing cells or
the bonding patter of striated muscles.
Types of Microscope

6. DARKFIELD MICROSCOPY
This utilizes oblique light that does not enter
the objective lens. A special darkfield is
employed and a vacant field of view shows
merely a dark background while the
objects appear bright.
 CARE FOR MICROSCOPE
Care of Lenses
• The microscope lenses should be cleaned with lens
paper before and after each use. Other material such
as laboratory tissue may scratch the lenses. It is
especially important that lenses never be left with oil
on them. Oil will soften the cement (glue) that holds
the lens in the objective.
 CARE FOR MICROSCOPE
Transporting the microscope
•A microscope should be left in a permanent
position on a study lab table in an area where
it will not get jammed. However, if the
microscope must be moved, it should be held
securely with one hand supporting the base
and the other hand holding the arm. The
microscope should be placed gently on tabletops,
to avoid jarring.
 CARE FOR MICROSCOPE
Storage of Microscope
• When the microscope is not being used, it
should be left with the low power
objective in position and the
nosepiece in the lowest position. The
stage should be centered so that it does
not project from either side of the
microscope. The microscope should be
stored in a plastic dust cover.
 PRECAUTIONS
• Use
the coarse adjustment only with the low
power objective
• Use oil each time the oil immersion lens is used
• Useimmersion oil with the oil immersion
objective only.
• Clean all oculars and objectives with lens paper
after each use.
• Moveor transport the microscope with one hand
under the base and the other hand gripping the
arm.
• Avoid jarring or bumping the microscope.
• Store the microscope covered in a safe area.