A TERM PAPER

ON

FIXATION

BY HUMAN ANATOMY
GROUP 1
SUBMITTED TO: DR. OLAWUYI
LIST OF GROUP MEMBERS

NAME MAT. NUMBER

1) ODEMAKINDE OLUWATOYOSI M. HUA/15/3272

2) AROWOSEGBE OLUKAYODE A. HUA/15/3247

3) ADEDOTUN OLUWAFEMI A. HUA/15/3220

4) OLUWABUSUYI BUKUNMI B. HUA/15/3285

5) GOLD NIFEMI T. HUA/15/3263

6) ANI CHUKWUEMEKA K. HUA/15/3245

7) ADEBIYI AYOBAMI M. HUA/15/3218

8) IFENATUOHA CHIBUZOR W. HUA/15/3265

9) OGUNJEMITE PONMILE I. HUA/15/3273

10) IJAGBEMI KAYODE O. HUA/15/3266

11) OLANIYAN HAMEEDAT O. HUA/15/3281

12) EHINAFE OLUWASEUN M. HUA/15/3255

13) EGABOR BLESSING O. HUA/15/3254

OUTLINE
 DEFINITION OF FIXATION

 PRINCIPLE OF FIXATION

 AIMS AND EFFECT OF FIXATION

 TYPES OF FIXATIVES

 EXAMPLES OF FIXATIVES

 PROCESS OF FIXATION

 FACTORS AFFECTING FIXATIVES

 PURPOSES OF FIXATION

 CONCLUSION
 FIXATION

*Introduction

In the fields of histology , pathology , and cell biology , fixation is a

critical step in the preparation of histological sections by which biological
tissues are preserved from decay, thereby preventing autolysis or
putrefaction . The structure of a tissue is determined by the shapes and
sizes of macromolecules in and around cells. The principal
macromolecules inside a cell are proteins and nucleic acids. Fixation
terminates any ongoing biochemical reactions, and may also increase the
mechanical strength or stability of the treated tissues. The broad objective
of tissue fixation is to preserve cells and tissue components and to do this
in such a way as to allow for the preparation of thin, stained sections.

*Definition

It is a complex series of chemical events which brings about changes

in the various chemical constituents of cell to preserve morphology and
structural detail.

*Principle of fixation

The fixative brings about cross linking of proteins which produces

denaturation or coagulation of proteins so that the semifluid state is
converted into semisolid state for easy manipulation of tissue.

*Aims and Effects of Fixation

1.To preserve the tissue as life-like as possible.
2.To prevent postmortem changes like autolysis and putrefaction.
3.Preservation of chemical compounds and micro-anatomic constituents
so that further histochemistry is possible.
4.Hardening : the hardening effect of fixatives allows easy manipulation
of soft tissue like brain, intestines etc.
5.Solidification: Converts the normal semifluid consistency of cells (gel)
to an irreversible semisolid consistency (solid).
6.Optical differentiation: it alters to varying degrees the refractive indices
of the various components of cells and tissues so that unstained
components are more easily visualized than when unfixed.
7.Effects of staining: certain fixatives like formaldehyde intensifies the
staining character of tissue especially with haematoxylin.

*Types of Fixatives

There are generally three types of fixation processes depending on the

initial specimen:
1) Heat fixation: After a smear has dried at room temperature, the
slide is gripped by tongs or a clothespin and passed through the flame of a
Bunsen burner several times to heat-kill and adhere the organism to the
slide. Routinely used with bacteria and archaea. Heat fixation generally
preserves overall morphology but not internal structures. Heat denatures
the proteolytic enzyme and prevents autolysis. Heat fixation cannot be
used in the capsular stain method as heat fixation will shrink or destroy
the capsule ( glycocalyx ) and cannot be seen in stains.

2) Immersion: The sample of tissue is immersed in fixative solution

of volume at a minimum of 20 times greater than the volume of the tissue
to be fixed. The fixative must diffuse through the tissue to fix, so tissue
size and density, as well as type of fixative must be considered. This is a
common technique for cellular applications. Using a larger sample means
it takes longer for the fixative to reach the deeper tissue.

3) Perfusion: Fixation via blood flow. The fixative is injected into

the heart with the injection volume matching cardiac output. The fixative
spreads through the entire body, and the tissue doesn't die until it is fixed.
This has the advantage of preserving perfect morphology, but the
disadvantages are that the subject dies and the cost of the volume of
fixative needed for larger organisms is high.

4) Chemical fixation: In both immersion and perfusion fixation

processes, chemical fixatives are used to preserve structures in a state
(both chemically and structurally) as close to living tissue In the fields of
histology , pathology , and cell biology , fixation is a critical step in the
preparation of histological sections by which biological tissues are
preserved from decay, thereby preventing autolysis or putrefaction . The
structure of a tissue is determined by the shapes and sizes of
macromolecules in and around cells. The principal macromolecules
inside a cell are proteins and nucleic acids. Fixation terminates any
ongoing biochemical reactions, and may also increase the mechanical
strength or stability of the treated tissues. The broad objective of tissue
fixation is to preserve cells and tissue components and to do this in such a
way as to allow for the preparation of thin, stained sections.

*Examples of Fixatives

1.Aldehydes: include formaldehyde (formalin) and glutaraldehyde.

Tissue is fixed by cross-linkages formed in the proteins, particularly
between lysine residues.
2.Mercurials: fix tissue by an unknown mechanism. They contain
mercuric chloride and include such well-known fixatives as B-5 and
Zenker's.
3.Alcohols: including methyl alcohol (methanol) and ethyl alcohol
(ethanol), are protein denaturants and are not used routinely for tissues
because they cause too much brittleness and hardness. However, they are
very good for cytologic smears because they act quickly and give good
nuclear detail.

4.Oxidizing agents: include permanganate fixatives (potassium

permanganate), dichromate fixatives (potassium dichromate), and
osmium tetroxide. They cross-link proteins, but cause extensive
denaturation.

5.Picrates: include fixatives with picric acid. Foremost among these is

Bouin's solution. It has an unknown mechanism of action.

6.Microwaves: MW irradiation may serve as the primary method of

fixation, MWs can also accelerate the fixing action (cross-linking) of
aldehydes or alcohols.

7.Vapour fixation: various chemicals which act as vapour fixatives

include aldehydes (formaldehyde, glutaraldehyde and acrolein), osmium
tetroxide, chromyl chloride, ethanol, diethylpyrocarbonate,
benzoquinone, and diacetyl; the most common being formaldehyde,
osmium tetroxide, and perhaps alcohol.

*Process

Fixation is usually the first stage in a multistep process to prepare a

sample of biological material for microscopy or other analysis. Therefore,
the choice of fixative and fixation protocol may depend on the additional
processing steps and final analyses that are planned. For example,
immunohistochemistry uses antibodies that bind to a specific protein
target. Prolonged fixation can chemically mask these targets and prevent
antibody binding. In these cases, a 'quick fix' method using cold
formalin for around 24 hours is typically used. Methanol (100%) can also
be used for quick fixation, and that time can vary depending on the
biological material. For example, MDA-MB 231 human breast cancer
cells can be fixed for only 3 minutes with cold methanol (-20 °C). For
enzyme localization studies, the tissues should either be pre-fixed lightly
only, or post-fixed after the enzyme activity product has formed.

*Factors Affecting Fixation

There are a number of factors that will affect the fixation process:
1.Buffering: there must be buffering capacity in the fixative to prevent
excessive acidity. Acidity favors formation of formalin-heme pigment
that appears as black, polarizable deposits in tissue
2.Penetration: Penetration of tissues depends upon the diffusability of
each individual fixative, which is a constant.
3.Volume: There should be a 10:1 ratio of fixative to tissue.

4.Temperature: increasing the temperature, as with all chemical

reactions, will increase the speed of fixation, as long as you don't cook
the tissue.
5.Concentration: Concentration of fixative should be adjusted down to
the lowest level possible, Too high a concentration may adversely affect
the tissues and produce artifacts similar to excessive heat.
6.Time interval: very important is the time interval from of removal of
tissues from the body to fixation. The faster you can get the tissue and fix

it, the better.

*Purposes
In performing their protective role, fixatives denature proteins by
coagulation, by forming additive compounds, or by a combination of
coagulation and additive processes. A compound that adds chemically to
macromolecules stabilizes structure most effectively if it is able to
combine with parts of two different macromolecules, an effect known as
cross-linking. Fixation of tissue is done for several reasons. One reason is
to kill the tissue so that postmortem decay (autolysis and putrefaction) is
prevented. [1] Fixation preserves a sample of biological material ( tissue
or cells ) as close to its natural state as possible in the process of
preparing tissue for examination. To achieve this, several conditions
usually must be met.
First, a fixative usually acts to disable intrinsic biomolecules—
particularly proteolytic enzymes —which otherwise digest or damage the
sample.
Second, a fixative typically protects a sample from extrinsic damage.
Fixatives are toxic to most common microorganisms (bacteria in
particular) that might exist in a tissue sample or which might otherwise
colonize the fixed tissue. In addition, many fixatives chemically alter the
fixed material to make it less palatable (either indigestible or toxic) to
opportunistic microorganisms.
Finally, fixatives often alter the cells or tissues on a molecular level to
increase their mechanical strength or stability. This increased strength and
rigidity can help preserve the
morphology (shape and structure) of the sample as it is processed for
further analysis.
Even the most careful fixation does alter the sample and introduce
artifacts that can interfere with interpretation of cellular ultra-structure. A
prominent example is the bacterial mesosome , which was thought to be
an organelle in gram-positive bacteria in the 1970s, but was later shown
by new techniques developed for electron microscopy to be simply an
artifact of chemical fixation. [2][3] Standardization of fixation and other
tissue processing procedures takes this introduction of artifacts into
account, by establishing what procedures introduce which kinds of
artifacts. Researchers who know what types of artifacts to expect with
each tissue type and processing technique can accurately interpret
sections with artifacts, or choose techniques that minimize artifacts in
areas of interest.

*Conclusion

Fixation is the most important process in tissue processing and it must

be properly done, because a poorly fixed tissue will putrefy/autolize and
give an inaccurate histologic/microscopic result and give a bad
morphology to the tissue while an over-fixed tissue will chemically mask
the target aspect and prevents antigen binding to the tissue.