Animal Histology

Course content
1. Fixatives and stains in animal histology
2. Animal histological techniques which include
Fixation,
Dehydration,
Clearing,
Embedding,
Sectioning,
Staining,
Mounting.
 Fixatives in animal histology

An important aspect of all histological and cytological techniques

is preservation of cells and tissues from degradation.

Therefore, fixatives are chemical substances or solutions

that preserves cell or tissue specimens. Fixatives change

the original chemical and physical compositions of tissues, as

well as cause physical changes to cellular and extracellular

components of cells and tissues in order to preserve the cells

In order to preserve cells and tissues, tissue blocks, sections or smears are usually immersed
in a fixative fluid. The basic function of fixatives include:
1. These fixatives are used to prevent autolysis (enzymatic digestion of cells by the action of
its own enzymes, particularly in dead cells) by inactivating lysosomal enzymes, and they
stabilize the fine structure of the cells and tissues. Autolysis is a common challenge in
enzyme-rich tissues which prevent proper staining for microscopic examination.

2. Moreover, fixatives also prevent putrefaction (decomposition or breakdown of organic

3. Fixatives also enhance the mechanical toughness of cells and tissues in order to resist
destruction, as well as withstand the successive treatment by various reagents treatment
(dehydration, embedding, and sectioning) with little or no damaging effect.

4. To help in subsequent staining (assisting in attachment of stains to the components of the

1. Length of fixation: the appropriate time of fixation varies with respect to the type of tissue being experimented. If
the duration of fixation is longer, it can lead to over-cross-linking. However, if the time period of fixation is short,
this will prevent proper chemical penetration, and as such, cross-linking will not occur.
2. Temperature: temperature can directly affect fixation and as such the rate of fixation is increased with increase in
temperature which in turn can increase the rate of autolysis. However, if the temperature is low or decrease, the
diffusion rate will similarly decrease which in turn decrease the penetration time. For electron microscopic studies,
0-4oC is regarded as ideal temperature. However, most fixation is considered appropriate at room temperature (20-
25oC).
3. Concentration: fixatives have different appropriate concentration. Thus, if the concentration of the fixing agent is
high, it can damage the tissue or cellular structure. However, if the concentration is low, the fixative agent may
require a prolonged time for fixation.
4. Size: if the sample size is large, it may be difficult for the fixative to penetrate and reach the deeper part of the
tissue and this in turn will lead to autolysis of epithelium. The most appropriate thickness size of any specimen for
fixation is 4-6mm to ensure complete penetration of fixatives.
5. Osmolarity or osmotic concentration: this is the concentration of a solution defined or expressed as the total number
of solute particles per litre. Thus, if the osmolarity of tissue is the same as that of fixative, there will be no swelling
or shrinkage of the tissue. However, if the osmotic concentration of the fixative is lower (hypotonic) than the cell
or tissue, there will be swelling. However, if the osmotic concentration of the fixative is higher (hypertonic) than
the cell or tissue, there will be shrinkage.
 List of various fixing agents
1. Formaldehyde or formalin: formaldehyde is a gas that is dissolved in water in order to
form formalin compound. Formalin is therefore the saturated solution of formaldehyde in
water. Formalin is the most commonly used fixative in pathology. Meanwhile 10%
formalin is most appropriate for many tissues preservation. A 100% formalin is equivalent
to 37-40% formaldehyde while 10% formalin is equivalent of 4% formalin. Formaldehyde
can react with molecules of the tissues to form a stable complex. It can react with nucleic
acids and proteins and thus can penetrate between nucleic acids and proteins thereby
forming a stabilized shell of nucleic acids and protein complex.
2. Glutaraldehyde: this preserves the ultrastructure of the tissue; therefore, it is
usually employed in electron microscopy studies. As a result of the poor penetration, and
overhardening properties, it is not employed as tissue fixatives in light microscopy. It is
quite hazardous since its exposure may lead to irritation of the respiratory tract, skin, and
even digestive tract.
3. Osmium tetroxide: Osmium tetroxide is type of fixative that is both soluble in water
and in nonpolar solvents. For electron microscopic studies, osmium tetroxide is used as
secondary fixative, and it also performs well as stain and imparts contrast when observed
under electron micro­scope. Osmium tetroxide is also helpful for staining of lip­ids in frozen
sections. It is observed that fixation by osmium tetroxide causes swelling in tissue, which
can be decreased by adding sodium chloride or calcium chloride to fixatives. Continued
exposure to osmium tetroxide vapours can cause deposition into cornea, which eventually
leads to blindness.
 List of various fixing agents
4. Mercuric chloride: It chiefly reacts with cysteine and also reacts with amines,
amides, sulfydryl groups, and ammonium salts, and results in tissue hardness. It also
acts as a strong protein coagulant. Nowadays, mercurial fixatives are not routinely used
except for fixation of hematopoietic tissues. They are toxic in nature and should not be
allowed to come in contact with metals. These fixatives have slow penetration capacity,
so the thickness of the specimens being fixed by mercuric fixatives should be thin.
5. Glyoxal: Glyoxal is considered as alternative fixative to formalin because it is a
dialdehyde in nature. It is a bifunctional aldehyde. Its individual alde­hyde groups are
potentially reactive, and also cross-links can be established. Glyoxal fixed tissues may
demonstrate precise cellular details.
6. Pictric acid: Picric acid is an example of a coagulant fixative. It forms picrates with
basic protein groups, which causes coagulation. Although picric acid is not able to fix
most car­bohydrates and lipids, picric acid is the most advised fixative to preserve
glycogen. Brighter staining is seen by picric acid fixatives.
7. Ethanol and methanol: For ethanol and methanol, fixation initiates at 50 to 60% concentration
and greater than 80% concentration, respec­tively. They are known to be coagulants that cause
protein denaturation. They cause interruption in hydrogen and hydrophobic bonding by substituting
water in tissue envi­ronment, which results in change in tertiary structure. Ethanol causes
mispresentation of cytoplasmic as well as nuclear details, but sometimes it can be used for
preservation of glycogen. Methanol is more commonly used for fixation of exfoliative cytology
smears and blood films.
 List of various fixing agents

8. Acetone: Acetone acts as an efficacious lipid solvent that results in tissue brit­tleness. Apart
from tissue fixation, they are primarily used as an agent for dehydration in tissue processing.

9. Acetic acid: Acetic acid is considered as a noncoagulated fixative agent. It acts by causing
nuclear proteins coagulation. Incidentally, it stabilizes and assists to prevent nucleic acids loss.
Acetic acid, when combined with ethanol, is used as an effective cytolog­ical fixative that helps
in conservation of nucleic acids, but if it is used singly, it results in swelling of cells. Time
required for fixation by acetic acid is less as penetration of acetic acid is faster into tissues.

10. Potassium dichromate: Potassium dichromate is also a noncoagulant fixative, but if used
in combination with acid solution, it acts as a coagulant fixative. It is seldom used alone for
fixation because chro­mate ions will link with few lipids and makes them insoluble.

11. Bouin’s fixative: Bouin’s fixative is considered as good fixative for conserving delicate as
well as soft tissue structures. The major portion of Bouin’s fixative contains picric acid with
little quantity of acetic acid as well as formaldehyde. It is considered toxic and are not allowed
in some laboratories. Other fixatives include acrolein and Genipin.
 Mechanisms of chemical fixation

The two major mechanisms of chemical fixation include:

1. Cross-linking: this involves the formation of covalent bond both within and
between proteins which lead to tissue toughness, thereby preventing
degradation.

2. Coagulation: this involves the dehydration of proteins by employing alcohol or