Joseph Haynes

Histology Assignment No.1

Histology and its methods of study; how do these methods differ from those of
Histopathology?

Student Name: Joseph Haynes

Bibliography: Histology and Cell Biology Examination and Board Review 5th edition, Douglas
F. Paulsen PhD, Janqueira’s Basic Histology Text & Atlas, Anthony L. Mescher

Histology is the study of the tissues in the body and is a largely visual science that relies on
microscopy and other imaging modalities to reveal cell, tissue and organ structure specifically
focusing on how cells’ structure and arrangement optimize functions specific to each organ.
Tissues are made of two interacting components: cells and extracellular matrix. The small size of
cells and matrix components makes histology dependent on the use of microscopes. Therefore
Histologic methods involve dividing tissues and organs in preparation for microscopic
examination and chemical analyses. The proceeding paragraphs will address the basic steps used
in tissue preparation for histology, the types of microscopy (light and electron microscopy) as
well as histochemical methods.

The basic methods of tissue preparation for microscopy include fixation, dehydration,
clearing, infiltration, embedding, sectioning, mounting and staining. If a permanent section is
desired, tissues must be fixed. Fixation preserves the structural organization of cells, tissues and
organs and prevents bacterial and enzymatic digestion (autolysis). It also insolubilizes
components to prevent diffusion and reduces damage in later steps in tissue processing. There
are two types of fixation which are chemical fixation and physical fixation or freezing. In
chemical fixation the tissues are usually immersed in solutions of stabilising or cross-linking
agents called fixatives. The tissues are usually cut into small fragments before fixation to
facilitate the penetration of the fixative to guarantee preservation of the tissue. Intravascular
perfusion of fixatives can be used and fixation is greatly improved since the fixative is pumped
through the blood vessels and rapidly reaches the tissues. One of the best fixatives used for
routine light microscopy is formalin, a buffered isotonic solution of 37% formaldehyde. As the
electron microscope affords high resolution, greater care in fixation is necessary to preserve
ultrastructural detail. Thus, a double fixation procedure using a buffered glutaraldehyde solution
(another widely used fixative) followed by a second fixation in buffered osmium tetroxide is a
standard procedure for fine structural studies. The osmium tetroxide preserves and stains lipids
and proteins. Limitations of chemical fixation include the fact that fixative-induced changes in
chemical composition and line structure may produce staining artifacts which are minor
structural abnormalities. Freezing is also used for light and electron microscopy where the tissue
is embedded in cryoprotectant (glycerine). Rapid freezing reduces ice crystal formation and
associated artifacts. This method allows tissue to be sectioned without dehydration or clearing
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thus it is faster and avoids dissolving lipids and denaturing proteins. However freezing is less
permanent, the sections are thicker and resolution is poor.

Embedding is usually preceded by two main steps: dehydration and clearing. The water is first
extracted from the fragments to be embedded by being immersed successively in a graded series
of mixtures of ethanol and water, usually from 70% to 100% ethanol. This is dehydration and
may denature proteins due to the ethanol. In addition water loss may cause uneven shrinkage of
components with different water content and create unnatural spaces between cells and tissue
layers. The ethanol is then replaced with a series of clearing-agent alcohol mixtures or a solvent
that is miscible with both alcohol and the embedding medium. As the tissues are infiltrated with
this solvent, they generally become transparent. Once the tissue is impregnated with the solvent,
it is placed in melted paraffin in an oven typically at 52-60 degrees Celsius. Xylene is a paraffin
solvent commonly used for light microscopy while Propylene oxide is a plastic solvent
commonly used for electron microscopy. 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 removal from the oven. This is Embedding. Tissues to be embedded with plastic
resin, usually for electron microscopy, are also dehydrated in ethanol and subsequently infiltrated
with plastic solvents, depending on the type of resin used 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 effect of the high temperatures needed for paraffin embedding
and gives little or no distortion to the cells. The harder embedding media allow thinner
sectioning which is a requirement for electron microscopy. The hard blocks containing the
tissues are then placed in an instrument called a microtome with a steel or glass blade that cuts 3
to 8 micrometre sections for light microscopy. Glass or diamond knives cut 1 to 5 micrometre
sections. Ultramicrotomes are also used for electron microscopy which cut very thin sections
(0.08 to 1 micrometre) with a glass or diamond knife. This is called sectioning. For tissues fixed
by freezing, sectioning is done by an instrument called a cryostat, a freezing microtome or
standard microtome in a refrigerated chamber. As previously mentioned, this method allows the
rapid preparation of sections thus it is routinely used in hospitals to study specimens during
surgical procedures. It is also effective in the histochemical study of very sensitive enzymes or
small molecules since it does not inactivate most enzymes.

Mounting eases handling and decreases damage to specimen during examination. For light
microscopy, sections are usually placed on glass slides, often pre-coated with thin layer of
albumin, gelatin or polylysine to improve attachment. After staining, sections are covered b glass
coverslips to preserve them for repeated examination. For electron microscopy, specimens are
mounted on copper grids since the electron beam cannot penetrate glass.

Most tissues are colourless and the substructure is indistinguishable therefore sections must be
typically stained in order to be studied microscopically. Methods of staining tissues have
therefore 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 selectively. Most
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stain affinities are based on reciprocal acid-base characteristics of stain and tissue components.
Most of these dyes behave like acidic or basic compounds and have a tendency to form
electrostatic (salt) linkages with ionisable radicals of the tissues. Tissue components with a net
negative charge stain more readily with basic dies and are termed basophilic; components with a
net positive charge such as proteins with many ionized amino groups, have affinity for acidic
dyes and are termed acidophilic. Some basic dyes include toluidine blue, alcian blue and,
methylene blue. Hematoxylin behaves like a basic die and stains the basophilic tissue
components. The main tissue components that ionize and react with basic dyes do so because of
acids in their composition. Acid dyes such as orange G, eosin and acid fuchsin, stain the
acidophilic components of tissues such as mitochondria, secretory granules and collage.

Of all dyes, the hematoxylin and eosin (H&E) combination is used most commonly and it also
distinguishes the nucleus from the cytoplasm.