Introduction to Histology

and Histological Techniques

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

Definition of Histology:

• “Histo” comes from Greek which means “web” or “tissue” and

“Logos” meaning “study”

• Therefore, histology is the study of tissues

• Histology is important in the understanding of many subjects of

Medical Science.
• Histology requires the use of “Microscopes” to view the
structures under increasing magnifications.

• This requires preparation of slides that will then be viewed

under the microscope.
Histological Techniques

• Involve preparation of the Tissues for Microscopic Examination

• The aim is to preserve the Microscopic Anatomy of the Tissue.

• The tissue is made somewhat hard so that very thin sections 4 to 5

Microns can be made.

• Good Staining is possible and required.

• Final stained Section closely represents a Live Tissue

Tissue Preparation

• The ideal microscope tissue preparation should be preserved so that the

tissue on the slide has the same structure and molecular composition as it
had in the body.

• This is sometimes possible but, as a practical matter, rarely feasible, and

artefacts, distortions, and loss of components due to the preparation process
are almost always present.

• There are about 6 steps involved in tissue preparation

1. Fixation
• If a permanent section is desired, tissues must be fixed.
• This is to avoid tissue digestion by enzymes present within
the cells (autolysis) or by bacteria and to preserve the
structure and molecular composition
• This treatment can be done by chemical or, less frequently,
physical methods.
• In chemical fixation, the tissues are usually immersed in
solutions of stabilizing or cross-linking agents called fixatives.
• One of the best fixatives for routine light microscopy is a
buffered isotonic solution of 4% formaldehyde.

• Formaldehyde and glutaraldehyde, another widely used

fixative, are known to react with the amine groups (NH2) of
tissue proteins.

• In view of the high resolution afforded by the electron

microscope, greater care in fixation is necessary to preserve
ultrastructural detail.
2. Dehydration and clearing

• Purpose: To remove all the water because the Paraffin

(embedding medium) is immiscible in water.

• Dehydration: The tissue is placed in increasing

concentrations of alcohol beginning with 70% to 100%.

• Each step takes about 2 to 3 hours.

• Clearing: This involves removing the alcohol and replacing it
with a chemical that is miscible in both alcohol and paraffin.

• The chemical is Xylene solution which will now infiltrate the

tissues.

• Smaller tissues take upto an hour.

• Larger ones require 2 to 4 hours.

3. Embedding

• To obtain thin sections with the microtome, tissues must be

infiltrated after fixation with embedding substances that impart a
rigid consistency to the tissue.

• Embedding materials include paraffin and plastic resins.

• Paraffin is used routinely for light microscopy; resins are used for
both light and electron microscopy.
• In paraffin embedding, the solvent used is usually xylene.

• Once the tissue is impregnated with the solvent, it is placed in

melted paraffin in the oven, typically at 58–60°C.

• The heat causes the solvent to evaporate, and the spaces

within the tissues become filled with paraffin.

• The tissue together with its impregnating paraffin hardens after

being taken out of the oven.
• Tissues to be embedded with plastic resin are also dehydrated in
ethanol and, depending on the kind of resin used, subsequently
infiltrated with plastic solvents.

• The ethanol or the solvents are later replaced by plastic solutions that
are hardened by means of cross-linking polymerizers.

• Plastic embedding prevents the shrinking caused by the high

temperatures needed for paraffin embedding and gives much better
results.
4. Sectioning

• The hard blocks containing the tissues are then taken to a

microtome and are sectioned by the microtome's steel or
glass blade to a thickness of 1–10 micrometres.
• A completely different way to prepare tissue sections is to
submit the tissues to rapid freezing.

• In this process, the tissues are fixed by freezing (physically,

not chemically) and at the same time become hard and thus
ready to be sectioned.

• A freezing microtome, the cryostat (Gr. kryos, cold, + statos,

standing), has been devised to section the frozen tissues.
• Because this method allows stained sections to be prepared rapidly
(within a few minutes), it is routinely used in hospitals to study
specimens during surgical procedures.

• Freezing of tissues is also effective in the histochemical study of very

sensitive enzymes or small molecules, since freezing does not
inactivate most enzymes.

• Because immersion of tissues in solvents such as xylene dissolves the

tissue lipids, the use of frozen sections is advised when these
compounds are to be studied
5. Mounting and Staining

• Mounting: the thin sections obtained from the microtome

are mounted upon glass slides.

• Staining: To be studied microscopically, most sections must

be stained.

• With few exceptions, most tissues are colorless, so observing

them unstained in the light microscope is useless.
• Methods of staining tissues have been devised that not only
make the various tissue components conspicuous but also
permit distinctions to be made between them.

• The dyes stain tissue components more or less selectively.

• Most of these dyes behave like acidic or basic compounds
and have a tendency to form electrostatic (salt) linkages with
ionizable radicals of the tissues.

• Tissue components that stain more readily with basic dyes

are termed basophilic (Gr. basis, base, + phileo, to love);
those with an affinity for acid dyes are termed acidophilic
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.
• 3. Fluorescence microscopy: used to view inherently fluorescent substances
or those tissues that have been labelled by fluorescent stains.

• Uses a light of a different wavelength (UV light), is focused onto the cells
which in turn emit light in the visible spectrum that can be viewed.

• 4. Confocal microscopy: a bright-field microscope uses large light source to

project light thus reducing the contrast in the image and hence poor
resolution.

• The confocal microscope therefore uses a small yet intense source of light, the
laser, and also a plate bearing a pin-hole aperture to reduce scattering of light
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.
Cells and tissue cultures

• Live cells and tissues can be maintained and studied outside

the body.

• In a complex organism, tissues and organs are formed by

several kinds of cells.

• These cells are bathed in blood plasma, which contains

hundreds of different molecules.
• Cell and tissue culture has been very helpful in isolating the
effect of a single molecule on one type of cell or tissue.

• It also allows the direct observation of the behavior of living

cells under a microscope.

• Several experiments that cannot be performed in the living

animal can be reproduced in vitro.
Cell fractionation

• Organelles and other components of cells and tissues can be

isolated by cell fractionation.

• This is the physical process by which centrifugal force is used

to separate organelles and cellular components as a function
of their sedimentation coefficients.
• The sedimentation coefficient of a particle depends on its
size, form, and density and on the viscosity of the medium.

• The organelles obtained with these techniques can be

analysed for purity in the electron microscope, and their
chemical composition and functions can be studied in vitro.
Histochemistry and Cytochemistry

• The terms histochemistry and cytochemistry are used to indicate methods for
localizing substances in tissue sections.

• Several procedures are used to obtain this type of information, most of them
based on specific chemical reactions or on high-affinity interactions between
macromolecules.

• These methods usually produce insoluble coloured or electron-dense

compounds that enable the localization of specific substances by means of
light or electron microscopy
End…