Part I

Last Update: 2 November 2017 Fixation M-5

Q: What is fixation? Describe why fixation of tissue is essential for histological study? What are the main aims of
fixation? Define fixative. What are the properties of fixative? What are the essential criteria of a good fixative?
Comment on the additive and non-additive fixatives. Give an example of fixative with its composition.
1+2+2+1+2+2+(3+3)

Many kind of cells can not be isolated for separate study while still a live. Such cells can only be examined in
permanent preparations. Tissue and cells need to be stabilized in structure firmly while being sliced. In fact they to be
fixed more or less in their living form before they are sectioned for subsequent examination.
What is fixation?
Fixation is the foremost prior step in the microtechnique that brings about the death of the cell in such a way
that the structure of the living cells is preserved with a addition of a minimum number of artefacts (chemicals). It is useful
in maintain the chemical composition of cells as intact as possible near the like condition.
Why fixation is necessary in microtechnique?
If a piece of tissue is cut out from a living or recently dead organism and no special care is taken to keep it alive
or maintain its structure, it will soon undergoes following marked changes.
1) If it left in air, it will loss water by evaporation and will shrink or it left in fluid it will swell or shrink depending
upon the osmotic concentration of the fluid but fixatives arrest the such type of changes.
2) Prevent the Bacterial and fungal attack. The bacteria and fungi secrete some enzymes, which act as an
autocatalytic agent, and break the protein architecture of the cell but fixations prevents the autocatalytic activity of the
enzymes.
3) In the cell, it self contain cell-organelles like lysosome which contain a groups of enzyme like trypsin, pepsin etc.
which are collectively known as cathepsin. The cathepsin attack on the protein which maintain the cellular architecture as
a result, dissolve their own protein constituents into most simpler from by breaking its structure.
To prevent all these facts fixatives is very much necessary for microtechnique.
Aim of fixations
1) Fixatives lead to hardness the tissue and give them a consistence form.
2) Fixative coagulates the tissue sap and prevents easily loss of diffusible substances.
3) It improves the optical differentiation of cell components by changing refractive index, thus increase their (cellular
components) visibility.
4) It prepares the tissue for staining.
5) It fortifies tissues against the harmful effects of various stages in preparation of sections.
Thus a fixation is a forward-looking process.
What is fixative?
The fixative is chemical substances which have antiautolytic activity and brings about the sudden death of the
cells or tissues in such a manner that their morphological organization and chemical composition is retained in a
condition almost identical to that of the life-like condition. eg. formaldehyde, acetic acid, picric acid, mercuric chloride,
chromic acid, osmium tetraoxide etc.
What are the properties of fixative?
Following are the main properties of the fixatives:
1) Antiautolytic activity
2) Prevent the cellular shrink, swell, dissolve or distort.
3) Anti-proteolytic activity.
4) Anti-pathogenic activity.

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 5) Fortifies the protein architecture.
6) Anti-dedicational
7) Prevent osmosis and evaporation.
8) Scissors off the terminal radicals of proteins and make it insoluble to solvent.
9) Maintain almost life-like condition of a call.
10) Increase cellular rigidity, which prevent disruption during the post fixational processing.
Characteristics of ideal fixative?
Ideal fixative is a certain chemical which have all the properties of fixatives in addition to it contain also some following
properties as,
1) Rapid diffusible activity towards the center of a section (high-penetrability)
2) Low molecular weight.
3) It can separate liquid phases from solid phases of protoplasm and helps in precipitation of protein without
damaging of protein architecture.
4) Maintain the optimum contrast of colour.
5) Highly lipophilic in nature but 100% insoluble in lipid.
6) All chemical constituents of a cell (protein, lipid etc) are insoluble in fixatives.
Classical fixatives
All the properties are not found in a fixatives so, to get good result of fixatives more then one of the fixatives are
used in a mixture which exhibit some more properties all together. Which designated as classical fixatives.
Primary Fixatives
The coagulant and non-coagulant types of fixatives are collectively known primary fixatives. Since each of them
has some merits and demerits so, it is unusual in practice to mix two or more of them together.
On the basis of the mechanism of the fixation these are also two types as additive and non-additive.
(1) Additive fixatives
It is a fixatives which not only remove bound water but they also form specific cross linkage in the main structure
of proteins and become an integral part of proteins. As for example:
(1) Formaldehyde (HCHO)
(2) Picric acid (C6H2 (NO3)3OH
(3) Mercuric Chloride (HgCl3)
a) Advantage
(1) The entire structure of protein is well stabilized, as they become an integral part of the proteins.
(2) They forms specific cross-linkage thus increase stability of the protein architecture.
b) Disadvantage
(1) Although additive fixatives form specific cross-linkage for protein stability but its (proteins) nature structure
slightly changes due to cross-links.
(2) Produce some artefacts due to cross-link formation.
(3) Nonadditive fixatives
It is a fixatives which can stabilized the protein structure by removing bound water but they do never become an
integral part of the protein, for example,
1) C2H5OH (ethanol)
2) CH3COCH3 (acetone)
3) CH3COOH (acetic acid)
4) K2Cr2O7 (Potassium di-cromate)
1) Advantage
1) Remove the bound water.

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 2) Stabilised the main structure of protein
2) Disadvantage
1) The entire structure is not well stabilised.
2) They never produced any cross linkage of the protein backbone.
Mode of action of fixatives
When a price of tissue is immerged in a fixative, cellular death does not occur instantly. After some time, cellular death
occurs due to anoxia, change of pH & enzymatic actions. In this time the fixatives penetrates
into the tissue by simple diffusion in such a way that the outer most cell of the tissue are first
killed & fix & penetrate layer by layer gradually towards centre. These processes so quickly
occur that the post-mortem alteration doses not occur. The rate of penetrations is determined
by the size of the block to be fixed. For rapidly penetrating fixatives, the dimensions will 3-5
mm2 but in slowly penetrative fixatives this is not exceed 2 mm2. When the fixatives penetrates
periphery to centre, it become diluted by the cellular H2O.

Mechanism of fixatives action

As soon as fixatives enters in to the intracellular environment through the lipid bilayer of the cell membrane
they first react upon the structure of macromolecules [proteins]. The protein molecule in the cells where they arranged or
lie in parallel conditions with the appropriate homologous amino acid forwards face to face. This particular sites are
subjected to action of fixatives [additive types ] to form various types of cross link.
When the homologous amino acid become face to face, then they are produce an unstable bond between them. In that
particular condition if we used proper fixatives that is readily incorporated with in the bond then ultimately the structure
will be much stabilized with the stronger bond.

NH2 NH2 NH2 NH2

Lysin Lysin
Cross
linkage Glysine Glysine

Lysin Lysin Glysin - glysin cross

linkage with dissociation
of H2O molecule

COOH COOH COOH COOH

Cross link form with the disassociation H2O molecule

(1) Formation of methylene bridge
Methylene bridge is formed by 2 successive step. In first step the amino group of the amino acids react with
formaldehyde (HCHO) to produce methylol (NHCH2OH). Then in the 2nd step methylol reacts with the another amino
group of amino acids to produce methylene bridge between 2 protein molecule with further removing 1 molecule of
H2O.

(2) Marcaptan Bridge

Mercuric chloride (HgCl2) which acts on sulpha hydril group (SH-), carboxyl (COOH) amino groups (NH 2) of
protein when came face to face then produce mercuric linkage between these groups (S-Hg-S) of two proteins molecules
is designated as Mercaptan bridge

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Some examples of fixatives used in laboratory:
Ethanol:
70%-100% ethanol is used either alone or mixture for the fixation of nucleus along with certain basic dyes. It
dose not precipitate proteins but dissolve lipids. It makes the cell hard but cause shrinkage.
Potassium dichromate:
It is generally used in mixture and percentage is 2-7%. It renders the protein insoluble in water and fixes lipids it
is used for the fixatives of chromosomes and other cytoplasmic structure.
Formaldehyde:
The reactions of HCHO with tissue protein are numerous and complex. It can bind with a number of different
functional groups of these proteins and form a bridge-like between the two protein molecules forming hydroxymethyle
compound (methylol).
RH+HCHO R- Fig. Formation of mythylene bridge
CH2 (OH) Methylene bridge

According to French and H H

H H
N N Amino acid Amino acid
Edsall (1945) the
N H C H N
H H H H
compound is reactive and
+ HCHO + H2O
cadences with further H-
atoms to from a
methylene bridge (-CH2 -)
COOH COOH COOH COOH
in a manner illustrated
bellow.
Protein
R-HCHOH+HR
R-HCH-R+H2O

Later by Kallen and Jenks (1960) an alternative possibility was suggested. They found the product of the
condensation of aqueous formaldehyde (HCHO) as methylene glycol with an aliphatic primary amine was a dehydroxy
methyle derivatives formed by the addition of a second molecule of formaldehyde to the initially formed hydroxymethyle
RNH2 + CH2(OH)2 R-NH CH2OH +H2O
Hydroxymethyle initially formed and then
RNH-CH2(OH) +CH2O RN(CH2OH) CH2OH
RNHCH2OH RNCH2+ H2O

In the last reaction loss of water form the intermediates which observed more slowly then the addition of
another molecules of HCHO
Uses: 4-10% is used in mixture for the fixatives of Golgi mitochondria and enzymes along with some basic stains.
Advantage: Prevent the
NH2 NH2 NH2 NH2 subsequent extra hardening of the
tissues with alcohol it fix protein
Cys SH HS Cys Cys SH Hg HS Cys molecules in such a way that no
+ 2HgCl2 + 4HCl ppt are found and form additive
Cys SH HS Cys Cys SH Hg HS Cys compound. Lipid may be
destroyed.
Cross linking by HCHO:
COOH COOH COOH COOH
The groups in amino acid
involved in the fixatives of proteins

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by formaldehydes are amino, imino, amino peptides, guanidyl, hydroxyl, carboxyl SH (salphalydri/thiol) and aromatic
rings.
Methyl bridge may be formed between two similar group say NH 2, NH2 & peptide or between NH2 &NH. This
HCHO produce many cross-linking between the proteins molecules.

Disadvantage:
1) Causes little shrinkage, 2) Takes much more time, 3) Gives of a unpleasant vapour that causes irritation of eyes and
respiratory epithelium.

GLUTARALDEHYDE
Like formaldehyde which acts through the formation of cross-links between protein end-groups, glutaraldehyde has also
been used extensively as an agent for protein-protein linkage and hence for fixation. An aqueous solution of
glutaraldehyde (glutaric dialdehyde) is a complex mixture at room temperature, consisting of approximately 4% free
aldehyde, 16% monohydrate, 9% dihydrate and 70% hemiacetal. Free glutaraldehyde may form polymers, or a
monohydrate and a dihydrate, which may cyclize to give a hemiacetal which in turn may also polymerise. Some favour
the polymer as the reactive species while others suggest that pure, monomeric, glutaraldehyde is the best fixative and
much less inhibitory to enzymes than is the mixed monomeric-polymeric product. The success of glutaraldehyde as a
cross-linking agent may also depend on the large range of different molecules present simultaneously in the fixation
solution.
When glutaraldehyde solutions are kept for long periods at ambient temperatures, there is a tendency for precipitates to
form and for aldehyde levels to fall so that some method of purification may be required. Glutaraldehyde may be
purified to the monomeric form by removing oligomers, polymers and other impurities through simple shaking with
barium carbonate, vacuum distillation or treatment with activated charcoal3 and chromatography on Sephadex G-10 has
produced equally good results. Vacuum distillation after prior treatment of commercial glutaraldehyde solutions with
sodium chloride and ethanol has become the most widely used technique for purification.4
There are many variations in the preparation of this fixative, including the percentage of glutaraldehyde, additives, and
buffers. Because of its low penetration, only small blocks of tissues (1-2 mm3) fix well at temperatures of 1-4C. The fixed
tissue specimen can be stored in buffer solution for many months.
The slow penetration, the requirement for cold temperature and the need for a storage medium, have greatly limited the
use of glutaraldehyde in histology. It is, however, the most widely used fixative for standard electron microscopy.
Other uses for glutaraldehyde all of which rely its cross-linking properties include the preparation of tissue xenografts,
particularly cardiac valves, chemical sterilisation and disinfection. Glutaraldehyde has an inhibitory effect on catalase 5
allowing the selective demonstration of the peroxidase activity of peroxisomes.
MISCELLANEOUS ALDEHYDES
Acrolein (acrylic aldehyde) is mainly used in the tanning industry. It produces more cross-links than formaldehyde under
optimal conditions but is unpleasant to use and unstable at alkaline pH levels. Acrolein has a tendency to polymerise into
disacryl, a solid plastic when exposed to light. It has been employed as a fixative for enzyme cytochemistry as labile
enzymes like glucose-6-phosphatase are retained in tissue fixed in 4% acrolein.
Glyoxal (ethanedial, diformyl), malonaldehyde (malonic dialdehyde), diacetyl (2,3-butanedione) and the polyaldehydes
are other aldehydes which have been infrequently employed for fixation, mostly for special situations, to retain specific
enzymes or proteins for histochemistry. In terms of effectiveness as cross-linking agents glutaraldehyde is the most
efficient although acrolein, when present in excess, is nearly as efficient and succinic dialdehyde is also comparable.
OTHER CROSS-RECTING FIXATIVES
Many other reagents are available for use as protein cross-linking fixatives although these are not widely employed in
histology.

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Chloro-s-triazides or cyanuric chloride has been used for the preservation of mucins in rat salivary glands 6 and for
immunofluorescence studies.7
Carbodiimides are compounds which react with a carboxyl and an amino group with elimination of water to give a
peptide and the corresponding urea. This reaction can be used for cross-linking soluble proteins, such as the linkage of
small peptides to larger proteins like serum albumin to provide complexes suitable as antigenic stimuli, but they are
seldom employed as fixatives.
Diisocyanates are used for introduction of fluorescent labels into proteins. Diazonium compounds as stabilised salts have
been used to reduce diffusion of soluble enzymes from tissue sections and salts such as Fast Garnet GBC, Fast Red B, and
Fast Blue R were effective in fixing an otherwise soluble aminopeptidase in rat kidney.8
Diimido esters react rapidly with proteins forming cross-links between amino groups to result in amidines which are
stable to acid hydrolysis. These esters have been used as fixatives for electron microscopy9-10 and
immunohistochemistry.11
Diethylpyrocarbonate is a compound consisting of diethyl oxydiformate and ethoxyformic anhydrate which is employed
for cold sterilisation particularly of alcoholic beverages. This
compound reacts with tryptophan to quench background CH2 CH2 CH2 CH2
O O
fluorescence induced by aromatic residues. It was first used as CH2 CH2 CH2 OS CH2
a vapour phase fixative for freeze-dried blocks to preserve O O
CH CH CH CH
Hexavalant
antigenic determinant sites for proteins and peptides. It has + OsO4
osmium
CH CH compound
also been proposed as a liquid phase fixative for small blocks, CH CH
O O
in phosphate buffer at pH 6.0, especially if its solubility is CH2 CH2 CH2 OS CH2
O O
improved with the addition of small quantities of ethanol. CH2 CH2
CH2 CH2
Maleimides, a number of N-substituted bismaleimides, L I P I D
L I P I D
synthesised as sulphydryl reagents, possess mild cross-linking
properties for proteins. Lastly, benzoquinone, a compound unsaturated ketone, reacts with amines, amino acids and
proteins to give various additional products. It has been used as a fixative for peptide antigens in various endocrine
tissues, in both vapour and liquid phase.11

Oxidising agents: metallic ions and complexes

Much less is known of how metallic ions and oxidising agents react with proteins.

Osmium tetra oxide:

The so-called osmic acid (OsO4) is widely used in the field of electron microscopy. The mechanism of reaction of
a tissue for the purpose of fixation with OsO4 is not clearly known. However, in general agreement the most important
tissue component involved here unsaturated lipids. Unsaturated lipids reduce OsO 4 with formation of a compound
containing hexavalant osmium.
OsO4 also reacts with protein and the main positive reaction with cystenine, triethanol amine. Oleic acid
methional, arginine, ornithine, lysine and ascorbic acid.
Use: It is fixatives and also stain and it use at 0.5 2%. It fixes the cytoplasm, fats, mitochondria, and Golgi etc.
Mercuric salt: The mercury add itself to the protein in the form of amino. According to Pearse 1980 Hg++ combines with
the acid groups of proteins specially carboxyl, hydroxyl and phosphoric acid of nucleoprotein. It has selective affinity with
sulphahydril (thiol ) groups. According to Hugher (950) the reaction is
R.-SH+HgCl2 RSGCl2 +HCl
R.-SHgCl+R.SH(R-S)2Hg +HCl.
The second reaction is little important in histology.

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Remark: According to Pearse (1980) and Hopwood (1972) in inmno cytochenistry of stable peptide (as insulin) is
actually improved by the addition of HgCl2 to formalin mixture.

Picric acid:
Advantage :
1) Good fixatives for connective tissue.
2) Present extra hardening of dehydrating agent.
3) Dose not shrink the tissue considerably.
Disadvantage:
1) dose not fix carbohydrate.
2) Never forma stable salt with mutation
Mixture fixatives:
1) Buins:
Buins is a fixative mixture in which both additive and non-additive fixatives are mixed in a particular proportion,
which can rectify the demerits of both fixatives and fixation become well.
Composition: Saturated solution of picric acid-75 ml + Formalin- 25 ml + Glacial acetic acid 5ml.
Time : Time is required for fixatives overnight to are month
Uses: It is used for fixatives of chromosomes. It causes precipitation.
Advantage: Of all proteins cause fixed the tissue. It does not cause excessive hardening of the tissue.
Disadvantage : 1) slow fixative.
2) Do not fix mucilated tissue
2) Zenkars fluid :
Composition : Distilled water..100 ml.
HgCl2.5 gm.
K2Cr2O7...2.5g.m.
Na2SO41g.m.
Glacial acetic acid ..1ml.
Schaudins fluid:
Composition :-
1) Saturated solution of HgCl2.66ml.
2) 100% ethyle alcohol..33 ml.
3) Glacial acetic acid 1 ml.
Time required :- 1-2 hrs.

3) Carnays fluids :
Composition:
1) 100% ethyle alcohol .60ml.
2) Chloroform ..30ml.
3) Glacial acetic acid .10ml.
Time required: 1-2 hrs or more.
Uses: It is widely used for fixation of chromosomes
and nucleic acid because it has got a combined
properties of ethanol and CH3COOH.

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