General Principles of Tissue Preparation and Staining
Introduction
The study of tissue structure relies on the preparation of tissue samples in ways that allow their
structural
details to be viewed at light or electron microscopic levels. First, there is the method of studying tissue
using the light microscope. Second, there is the study of what is sometimes called the “ultrastructure”
of tissue using the transmission electron microscope. There are numerous variations on these two gen-
eral themes. Recognizing that a survey of all tissue preparation techniques is well beyond the scope of
this atlas, the general features of the two methods mentioned are briefl
y summarized here.
Preservation versus Fixation
Preservation and ± xation of tissues, for subsequent treatments, share to varying degrees the common
goals of minimizing any further tissue degradation and preserving the various components of the tissue
in as lifelike a condition as possible. While the terms
preservation
and
± xation
are frequently used as
interchangeable, they are, in fact, slightly different.
Preservation of tissue
accomplishes the ±
rst goal stated above (prevents further degradation) but
not necessarily the second. For example, a can of green peas contains preservatives; these may even be
listed on the label. They protect the peas from further degradation. If you take a pea out of the can and
look at it microscopically, its structure might be recognizable, but it is not very lifelike. Fish that are
preserved in salt undergo signi± cant changes that render the tissue useless for microscopic examination.
Such structures are preserved but are not ±
xed. Many of the substances used to preserve tissues (animal
and plant) can be ingested in moderation and cause absolutely no harm.
Fixation
,
on the other hand, protects against further degradation while preserving the internal
components of the cell in a strikingly lifelike appearance. Fixation also hardens the tissue, making it
possible to further manipulate the sample without damage. Tissue that is ±
xed will appear quite life-
like when viewed with a microscope. In this respect, a sample of tissue that is ±
xed is also preserved;
however, a sample of tissue that is preserved is not necessarily ±
xed. Another important difference is
the fact that most substances (except alcohol) used to ±
x tissue generally cannot be ingested; to do so,
even in very moderate amounts, would cause signi±
cant harm or death.
Fixatives and Methods of Fixation
Fixatives
In addition to preventing tissue degradation, preserving components of the tissue, and hardening the
tissue, proper ± xation will also transform the contents of the cell from a semifl
uid to a semisolid and
prepare the cell contents for visualization with stains, dyes, or metallic salts. There are numerous ±
xa-
tives, many of which are designed for unique applications, which can be used separately or in combina-
tions. Only representative examples are mentioned here.
Formalin
(mixture of formaldehyde and alcohol), in solution of 5% to 10%, is one of the more
commonly used general ±
xatives. It penetrates the tissue rapidly, leaves no residues, and requires little
or no washing of the tissue to remove. It is used alone, in a solution buffered with sodium phosphate
salts, or in other combinations. Buffered neutral formalin is usually preferred. Formalin acts through
cross-linking between proteins.
Picric acid
, in combination with formalin and glacial acetic acid (Bouin solution), is also used as
a ± xative. Its action of ± xation is not fully understood, and it does not harden the tissue as much as
formalin. Details of the nucleus are well demonstrated. However, picric acid in its dry form must be
handled with care because it is an explosion hazard. Picric acid can also be used as a stain.
Aldehydes
, such as glutaraldehyde and paraformaldehyde, are excellent ±
xatives for light micro-
scopic applications and are also widely used in electron microscopy. Aldehydes act rapidly but tend to
penetrate the tissue slowly. However, they provide excellent cellular detail regarding the contents of the
Appendix
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