INTRODUCTION TO

HISTOLOGICAL STUDIES
 INTRODUCTION
• Histology is the study of Cells, Tissues and
Organs as seen under the Microscope.
• Originally, it is the study of Tissues
• Histos: from the Greek word meaning tissue
and
• Logia means “Study of” or “Knowledge”.
 INTRODUCTION CON’T
• Histology is the science of the tissues.
• It studies Structures as well as Functions
• Knowing the function can predict the
structures
• Knowledge of the normal is necessary to
study the abnormal i.e. Pathology which
deals with the alterations in the structure &
function of the body, its organs, tissues and
cells caused by diseases
 TWO IMPORTANT CONSIDERATIONS
GIVEN WITH RESPECT TO THE
METHODOLOGY
1) The kind of Microscope used
2) The type of techniques used in preparing the
tissues suitable for viewing under the
microscope
 MICROSCOPY: BRIEF HISTORY OF
MICROSCOPES
• Robert Hooke and Marcello Malpighi in the
17th Century employed Simple Lenses to
study various Structural features.
• Between 1673 &1716: Compound
Microscope was developed by Leeuwenhoek
used in observing Protozoa, Bacteria,
Muscles, Nerves and many structures.
 HISTORY CON’T
• 18th Century saw slow development
• Early 19th Century Compound microscopes
become highly developed.
• Robert Brown discovered the Nucleus in
1831, in 1838, Shleiden and Schwann in
1839, Enunciated (state clearly) the cell
theory while in 1841, Henle Published the
1st comprehensive account of Human
Histology.
 HISTORY CON’T
• Though Bichat in 1802 described the Human
body as made of group of cells (tissues), but it
was Virchow in 1863 that described the
Human body as a “Cell State” and listed
specialized cell types. By the end of 19th
Century, Microtomes were developed
commercially with the development of fixing,
embedding & staining techniques continuing.
 TYPES OF MICROSCOPES
• Microscopes are classified based on the Light
source used
• The importance of any microscope depends
upon it’s
1.Ability to magnify structures
2.Resolve details: Resolution power is a
measure of the capacity of the microscope to
separate clearly two points close together
 TYPES OF MICROSCOPES CON’T
• Light or Optical Microscope: These are two (2) stage
magnifying device. An objective lens provides the initial
magnification and the ocular lens magnify the primary image
a second time. An additional condensing lens is present
beneath the stage to concentrate the light from the source
into a very bright beam.
• Polarizing Microscope: Developed to study
crystalline materials by mineralogists. Natural
objects exhibit optical properties called double
Refraction or Birefringence(the splitting of one ray of
light into two in an anisotropic medium). In
histological materials, birefringence is caused by the
orientation of particles small to be resolved. The
examination of birefringence permits deductions to
be made concerning the organization of structure
not shown by the light microscopes
• Phase Contrast Microscope: Makes the use of
Refractive indices of cytoplasm and its
inclusions are similar.
• Interference Microscope: Depends on the
ability of an object to retard light. This
depends on the ability to send through the
specimen two separate beams of light which
then are combined in the image plane.
 TYPES OF MICROSCOPES CON’T
• Dark field Microscope:
• Ultra Violet or Fluorescence Microscope
• X-ray Microscope
• Electron Microscope: Transmission & Scanning
EM
 STEPS IN TISSUE PROCESSING
• Fixation
• Dehydration
• Clearing
• Penetration
• Embedding
• Sectioning
• staining
STAGES OF TISSUE PROCESSING
 TISSUE PROCESSING
• Tissue fixation: by chemical e.g. formalin or
freezing with liquid Nitrogen.
• Dehydration: Dehydrate by passing through
graded levels of alcohol because paraffin is
not water soluble.
 TISSUE PROCESSING CON’T
• Clearing: Clear to remove the alcohol &
replace by the embedding medium using
clearing agents e.g. Xylene, Chloroform,
Benzene and Cedar wood oil.
• Penetration: Infiltration using embedding
agents which solidify e. g Paraffin Wax or
Celloidin
 TISSUE PROCESSING CON’T
• Embedding: Embed using Paraffin wax
• Sectioning: using microtome between 3 to 10
microme with egg albumen to mount sections
on glass slide
PHOTOGRAPH OF A MICROTOME &
EMBEDDED TISSUES
• Staining : this is done in aqueous solution e.g.
Hematoxylin & Eosin or specialized stains e.g.
Periodic Acid Schiff (PAS)
STAGES OF TISSUE PROCESSING
EXAMINATION & INTERPRETATION OF
TISSUE SECTIONS
• Require skills which need to be developed
• Reconstruct a 3-dimensional mental picture of
cells, tissues and organs from the 2-
dimensional sections
• Plane of section must be in mind e.g Cross
section or Longitudinal section
• What type of stain: General stains e.g.
Hematoxylin & Eosin(H&E) or Specialized
stains e.g. PAS
• Techniques used in preparation: Fresh or fixed
tissue preparation
• Any artifacts present: False staining or
preparation Due to preparation, staining,
sectioning methods, Shrinkage or wrinkling
• Type of Microscope used in viewing the tissue