HISTOLOGY AND ITS METHOD

OF STUDY

Mr Mickey Banda, Assistant Prof.

College of Medicine
Anatomy Department
Texila American University
 Objectives

• Students should be able to describe;

• The basic histological techniques

• Step by step tissue preparation

• Different types of tissue dyes and their uses

• The microscope and its different parts

• Other forms of tissue preparation and examination

 Introduction
• Histology - miscroscopic study of body tissues.

• The Greek root histo can be translated as

either “tissue”
• Histology requires the use of “Microscopes” to
view the structures under increasing
magnifications
 Cont`

• Tissues are usually webs of interwoven

filaments and fibers, both cellular and non-
cellular, with membranous linings.
• Tissues have two interacting components:
cells and extracellular matrix (ECM)
• ECM supports cells and cells produce ECM
 Units of measurements
For Light microscope
• The term micrometer (μm) is being used
nowadays instead of micron (μ).
• 1 micrometer or micron = 0.001 mm
For Electron Microscopy
• The term nanometer (nm) is being used
nowadays instead of angstrom (A°).
• 1 nanometer = 0.001 μm
Processing of Tissues for light microscope

Tissue fixation and Fixatives

• Chemical substances like formalin, mercuric
chloride, acetic acid, picric acid, ethanol,
methanol and glutaraldehyde are used as
fixatives to preserve tissues.
 Purpose of fixation
• to preserve the morphology and chemical
composition of the tissue
• to prevent autolysis and putrefaction (decay
or decomposition)
• to harden the tissue for easy manipulation
• to solidify colloidal material
• to influence staining
 Dehydration and clearing
• Purpose: To remove all the water

• Dehydration: The tissue is placed in increasing

concentrations of alcohol beginning with 70%
to 100% (ascending grades).
 Clearing
• After dehydration the tissue is treated with a
paraffin solvent, (clearing agent) like xylene,
toluene, chloroform, benzene, or petrol
• These agents penetrate and replace the
alcohol from the tissue and make it
translucent (clear).
 Embedding

• In order to obtain thin sections with microtome,

tissue is infiltrated with embedding medium
which gives a rigid consistency to the tissue.
• The various embedding media are paraffin wax,,
gelatin, plastic resins etc.
• Paraffin is the routinely used embedding medium
for light microscopy.
 Embedding involves two steps:
A. Impregnation
• After clearing, the tissue is impregnated with
molten paraffin wax
• The melting point of paraffin wax is 56 °C.
 Cont`

B. Casting or block making

• After impregnation, the tissue is placed in ‘L’
moulds containing molten paraffin.
• The molten wax cube with the tissue is
allowed to cool and the paraffin block is then
removed from the mould.
 Section Cutting (Microtome)
• 5–7 μm-thick sections are cut with a
microtome.
• The cut paraffin sections are affixed to glass
microslides after flattening the sections over
warm water.
• The microslides with sections are either air
dried or dried in an incubator overnight at 37
°C and stored for staining at room
temperature.
 Staining Procedure
• Use a basic and acidic dye that stain tissue
components selectively.
• Tissue components that stain with basic dyes
are termed basophilic and are blue in colour
• Tissue components with an affinity for acid
dyes are termed acidophilic and are
pink/orange in colour.
 Cont`
• Basic dyes: haematoxylin,
toluidine blue, alcian blue and
methylene blue.
• Acidic dyes: eosin, orange G and
acid fuschin
• Combination of haematoxylin
and eosin (H&E) is most
commonly used in histological
staining procedure.
 SPECIAL STAINS

• Periodic acid Schiff

reagent (PAS) –
• used to detects
polysaccharides such as
glycogen, glycolipids and
mucins in tissues. Stains
glycogen red
 SPECIAL STAINS
• Osmic Acid (Osmium
Tetroxide) Stain – used
to stain lipids – stain
black
• Aldehyde Fuchsin Aldehyde Fuchsin
• Used to pancreatic islet beta cell
granules
• Stains elastic fibers purple/black

• Cresyl Violet Stain (Nissl Stain)

• Stains both neurons and glia
• Stains cell bodies blue/violet
• Masson’s Trichrome Thin skin
• Trichrome means technique uses 3
colors
• Nuclei and other structures are
Stained blue
• Cytoplasm, muscle, rbc & keratin
are stained Bright red
• Collagen is stained Green or blue
Alcian blue
• stains mucin blue
• Cartilage is stained blue
Van Gieson
• Stains collagen red,
nuclei blue and rbc &
cytoplasm yellow
• Combined with an
elastin stain, stains it
blue/black
• Used for blood vessels
and skin
Sudan Black and Osmium
• Stains lipids in cells such as
myelin a brownish/ black
color

Reticulin (silver) stain

• stains reticular fibers blue
or black

• Golgi Stain – stains

neurons
Azan stain
• Nuclei are stained bright red, collagen, bm and
mucin are stained blue
• Muscle and rbcs are stained orange or red
• Good for staining ct and epithelium
• Stains - Fibrous connective tissue, mucus, and
hyaline cartilage
 Cont`
Wright`s stain
• Neutral stain produced by the interaction of an
acid and basic dye
• Basophilia – affinity for methylene blue
• Azurophilia – reddish blue
• Acidophilia – eosin
• Neutrophilia – pale lilac
• RBC – pink, nuclei – purplish blue, eosinophilic
granule – red, neutrophilic granules – reddish
brown.

Giemsa stain – stains blood cells, bone marrow

smears.
• Nuclei are stained dark-blue to violet
• Cytoplasm pale blue
• Rbcs pale pink
NB: A & C = Giemsa, B = Wright
Toluidine blue
• Stains acidic components various shades of blue
• Useful blue cationic dye
• Used for thin acrylic or epoxy sections
• Blood smear – neutrophils stain blue

Gomori Trichrome – mixture of 3 dyes. Stains CT

and collagen green or blue. Stains muscle,
keratin and cytoplasm red. Nuclei will stain
gray/blue/black
Silver and Gold Methods – used to demonstrate
fine structures such as cell processes in
nerves.
• Stains black, brown or golden stain
 Microscope
Types of Light Microscopes:
1. Compound/Bright-field/Light microscopy: Widely
used by histology students.
– Involves use of fixed and then stained slides to view under
an ordinary light.

2. Phase contrast microscopy: uses modified objective

lenses and condenser to allow the viewing of living
tissue without prior fixing or staining.
 Parts of a Microscope
• Eyepiece: The lens through which the viewer looks at the
specimen. Magnifies image 10X.
• Body tube (Head): Connects the eyepiece to the objective
lenses.
• Arm: Connects the body tube to the base of the microscope.

• Nosepiece: A rotating disc that bears objective lenses of

varying magnifications.
• Objective lenses: Used to magnify the specimen. A standard

microscope has objective lenses of 4X , 10X, 40X upto 100X.

• Stage: The flat platform where the slide is placed.

• Aperture: The hole in the center of the stage that allows light to

reach the specimen.

• Stage clips: Metal clips that hold the slide in place.

• Iris diaphragm: Adjusts the amount of light that reaches the

specimen.

• Coarse adjustment: moves the stage up and down in greater

increments.
• Fine adjustment: Fine tunes the focus by the moving the
stage in smaller increments.
• Stage Control: Moves the stage left and right.

• Condenser: Collects and focuses light from the illuminator

onto the specimen.
• Illumination: The light source for a microscope.

• Base: Supports the microscope and bears the illumination.

• On/off switch: Switch on the base of the microscope to

turn the light source on and off.
 The End
Thank you