Types of Description Classification Advantages Disadvantage Specific Use

Fixatives

Fumes are immunohistochemical

SIMPLE fixes the tissues by Aldehydes Formalin - It is irritating to the techniques.
FIXATIVE: forming cheap, readily nose and eyes
cross-linkages in Formaldehyde available, easy and may cause
Aldehydes sinusitis,
the proteins, to prepare, and
allergic rhinitis,
particularly relatively
or excessive
stable, lacrimation.
between lysine
residues. This especially if
cross-linkage does stored in
not harm the buffered
solution.
structure of
proteins greatly, so
that antigenicity is
not lost

10% 10%
Formal-Saline This is a simple Formal-Saline It penetrates It is a slow It is recommended for
microanatomical and fixes fixative. The fixation of central
fixative made up tissues evenly. period of nervous tissues and
of saturated fixation is general post-mortem
formaldehyde It preserves required to be
(40%, by weight microanatomic 24 hours or tissues for
volume) diluted to and cytologic longer. histochemical
10% with sodium details with examination. It is also
chloride. minimum recommended for the
shrinkage and preservation of lipids,
distortion. especially
phospholipids

Formol-Corrosi Formol-Corrosi It penetrates

ve Sat. Aq. Mercuric ve small pieces of Penetration is Formol-mercuric
(Formol-Subli chloride 90 ml. (Formol-Subli tissues rapidly. slow; hence, chloride solution is
mate) Formaldehyde mate) tissue sections recommended for
40% 10 ml. should not be routine post-mortem
more than 1 cm tissues
thick.
Glutaraldehyde Another popular it causes rapid It is more
aldehyde for and irreversible expensive.
fixation is changes, fixes It is less
glutaraldehyde, quickly, is well stable.
made up of two suited for
formaldehyde electron
residues, linked by microscopy, it
a three carbon fixes well at 4
chain. oC, and it gives
Glutaraldehyde is a best overall
larger molecule cytoplasmic
than formaldehyde, and nuclear
and so its rate of detail.
diffusion across
membranes is 1. It has a more
slower than stable effect on
formaldehyde. tissues, giving
a firmer texture
with better
tissue sections,
especially of
central nervous
tissues.

METALLIC Mercuric chloride It penetrates It causes

FIXATIVES: is the most and hardens marked
Mercuric common metallic tissues rapidly shrinkage of
Chloride fixative, frequently and well. cells (this may
used in saturated 2. Nuclear be counteracted
aqueous solutions components are by addition of
of 5-7%. Mercuric shown in fine acid).
chloride is widely detail.
used as a 3. It
secondary fixative precipitates all
reacting with a proteins.
number of amino
acid residues and
accompanied by
spectroscopic
changes, probably
due to reaction
with histidine
residues.

Zenker's Mercuric chloride It produces a Penetration is

Solution 5 gm Potassium fairly rapid and poor.
dichromate 2.5 gm even fixation of It is not stable
Distilled water 100 tissues. after addition
 ml Acetic acid, Stock of acetic acid.
glacial 5 ml (to be solutions keep Prolonged
added just before well without fixation (for
use) Heat, cool, disintegration. more than 24
filter in brown hours) will
bottle. Wash make tissues
sample for 24 brittle and
hours with distilled hard.
water after
fixation. Fixation
time: 12-24 hours

Zenker-Formol It is an Disadvantages: Zenker’s solution is

(Helly’s) excellent The an excellent fixative
Solution microanatomic disadvantages for bone marrow,
fixative for of Helly's extramedullary
pituitary gland, solution are hematopoiesis and
bone marrow similar to intercalated discs of
and blood Zenker's except cardiac muscle.
containing that brown
organs such as pigments are
spleen and produced if
liver. tissues
(especially
blood
containing
organs) are
allowed to stay
in the fixative
for more than
24 hours due to
RBC lysis.

Heidenhain's It penetrates Prolonged – is recommended

Susa Solution and fixes fixation of mainly for tumor
tissues rapidly thick materials biopsies especially of
and evenly. 2. may produce the skin; it is an
It produces considerable excellent cytologic
minimum shrinkage, fixative.
shrinkage and hardening and
hardening of bleaching;
tissues due to hence, tissues
the should not be
counter-balanc more than 1
e of the cm. thick.
swelling effects
of acids and the
shrinkage
effect of
mercury
CHROMATE - is used in 1-2%
FIXATIVES: aqueous solution,
Chromic Acid usually as a
constituent of a
compound fixative.
It precipitates all
proteins and
adequately
preserves
carbohydrates.

Potassium It fixes but does

Dichromate not precipitate
cytoplasmic
structures.
2. It preserves
lipids.
3. It preserves
mitochondria

Regaud’s 1. It penetrates 1. It
(Muller's) Fluid tissues well. deteriorates
2. It hardens and darkens on
tissues better standing due to
and more acidity; hence,
rapidly than the solution
Orth's fluid. must always be
3. It is freshly
recommended prepared.
for the 2. Penetration
demonstration is slow, hence,
of chromatin, tissues should
mitochondria, not be thicker
mitotic figures, than 2-3 mm.
Golgi bodies,
RBC and
colloid-containi
ng tissues.

Orth's Fluid It is Same as in

recommended Regaud's fluid.
for study of
early
degenerative
processes and
tissue necrosis.
2. It
demonstrates
rickettsiae and
 other bacteria.
3. It preserves
myelin better
than buffered
formalin.

LEAD Lead fixatives are 1. It is It takes up C02

FIXATIVES used in 4% recommended to form
aqueous solution for acid insoluble lead
of basic lead mucopolysacch carbonate
acetate. Lead arides. 2. It especially on
oxaloacetate, a fixes prolonged
primary reaction connective standing. This
product precipitate tissue mucin. may be
for the removed by
visualization of the filtration or by
activity of adding acetic
glutamic acid drop by
oxaloacetic drop to lower
transaminase in the pH and
tissue sections, is dissolve the
stable at a slightly residue
alkaline pH.

Osmium This is a pale OXIDIZING 1 It fixes It is very Osmium tetroxide is

Tetroxide yellow powder AGENTS conjugated fats expensive. traditionally used in
(Osmic Acid; which dissolves in and lipids 2. It is a poor electron microscopy
OsO4) water (up to 6% at permanently by penetrating both as a fixative and
20°C) to form a making them agent, suitable a heavy metal stain.
strong oxidizing insoluble only for small Osmium tetroxide is a
solution. Fixation during pieces of good fixative and
with osmium subsequent tissues (2-3 excellent stain for
tetroxide or treatment with mm. thick). lipids in membranous
postosmication of alcohol and 3. It is readily structures and
glutaraldehyde-fix xylene. Fats reduced by vesicles.
ed tissue causes form hydrated contact with
the complete osmium organic matter
denaturation of dioxide, are and exposure to
protein. stained black sunlight,
and therefore forming a black
are easier to precipitate
identify. which settles at
2. It preserves the bottom of
cytoplasmic the container.
structures well,
e.g. Golgi
bodies and
mitochondria.

Flemming's is the most It is an It is a poor

Solution common excellent penetrating
chrome-osmium fixative for agent; hence, is
acetic acid fixative nuclear applicable only
used, structures, e.g. to small pieces
recommended for chromosomes. of tissues.
nuclear preparation 2. It 2. The solution
of such sections. permanently deteriorates
fixes fat. 3. rapidly and
Relatively less must be
amount of prepared
solution is immediately
required for before use.
fixation (less
than 10 times
the volume of
the tissues to
be fixed).

Flemming's is made up only of same as

solution chromic and osmic Flemming's
without acetic acid, solution.
acid recommended for
cytoplasmic
structures
particularly the
mitochondria. The
removal of acetic
acid from the
formula serves to
improve the
cytoplasmic detail
of the cell.

PRECIPITATI 1. It is 1. Penetration
NG excellent for is slow.
(ALCOHOLIC fixing dry and 2. If left in
) FIXATIVES wet smears, fixative for
blood smears more than 48
Methyl and bone hours, t issues
Alcohol 100% marrow tissues. may be over
2. It fixes and hardened and
dehydrates at difficult to cut.
the same time.

Ethyl Alcohol - is used at 1. It preserves 1. Hemosiderin

concentrations of but does not fix preservation is
70-100%. If the glycogen. less than in
lower 2. It fixes buffered
concentrations are blood, tissue formaldehyde.
used, the RBC's films and 2. It is a strong
 become hemolyzed smears. reducing agent;
and WBC's are 3. It preserves hence, should
inadequately nucleoproteins not be mixed
preserved. It may and nucleic with chromic
be used as a simple acids, hence, is acid, potassium
fixative. It is, used for dichromate and
however, more histochemistry, osmium
frequently especially for tetroxide which
incorporated into enzyme are strong
compound studies. oxidizing
fixatives for better agents.
results. 3. Lower
concentration
(70-80%) will
cause RBC
hemolysis and
inadequately
preserve
leukocytes.

Newcomer's FORMULA: 1. It is
Fluid Isopropyl alcohol recommended
60 ml. Propionic for fixing
acid 30 ml. mucopolysacch
Petroleum 30 ml. arides and
Ether 10 ml. nuclear
Acetone 10 ml. proteins.
Dioxane 10 ml. 2. It produces
Fixation time: better reaction
12-18 hours at 3°C in Feulgen
stain than
Carnoy's fluid.
3. It acts both
as a nuclear
and
histochemical
fixative.

Gendre's Post-fixation with PRECIPITATI 1. Fixation is 1. It produces

Fixative phenol-formalin NG faster (fixation gross
for 6 hours or more (ALCOHOLIC) time is reduced hardening of
can enhance FIXATIVES to one-half). 2. tissues.
immunoperoxidase It can be used 2. It causes
studies on the for rapid partial lysis of
tissues, and in diagnosis RBC.
some cases, because it fixes
electron and dehydrates
microscopy, if it is at the same
necessary at a later time, e.g., in
time to establish a the frozen
 diagnosis. section room.

Carnoy’s FORMULA: It is considered 1. It produces

Fixative Absolute alcohol to be the most RBC
60 ml. Chloroform rapid fixative hemolysis,
30 ml. Glacial and may be dissolves lipids
acetic acid 10 ml. used for urgent and can
Fixation Time: 1-3 biopsy produce
hours specimens for excessive
paraffin hardening and
processing shrinkage. 2. It
within 5 hours. causes
2. It fixes and considerable
dehydrates at tissue
the same time. shrinkage. 3. It
3. It permits is suitable only
good nuclear for small pieces
staining and of tissues due
differentiation. to slow
penetration.

Bouin's The PICRIC ACID 1. It is an 1. It causes Bouin's Solution is

Solution complementary FIXATIVES excellent RBC hemolysis recommended for
effects of the three fixative for and reduces the fixation of embryos
ingredients of glycogen amount of and pituitary biopsies.
Bouin’s solution demonstration. demonstrable
work well together 2. It penetrates ferric iron in
to maintain tissues well and tissue.
morphology. fixes small 2. It is not
Specimens are tissues rapidly. suitable for
usually fixed in frozen sections
Bouin’s solution because it
for 24 hours. causes frozen
Prolonged storage sections to
in this acidic crumble when
mixture causes cut.
hydrolysis and loss
of stainable DNA
and RNA.

Brasil's PICRIC ACID 1. It is better

Alcoholic FIXATIVES and less
Picroformol "messy" than
Fixative Bouin's
solution. 2. It is
an excellent
fixative for
glycogen.

Compound -are those that

Fixatives: permit the general
microscopic study
of tissue structures
Microanatomi without altering
cal Fixatives the structural
pattern and normal
intercellular
relationship of the
tissues in question.

Cytological are those that

Fixatives: preserve specific
parts and particular
Nuclear microscopic
Fixatives: elements of the
cell itself.
Flemming's
fluid Carnoy's
fluid Bouin's
fluid
Newcomer's
fluid
Heidenhain's
Susa

Cytoplasmic are those that

Fixatives: preserve
cytoplasmic
Flemming's structures in
fluid without particular. They
acetic acid must never contain
Kelly's fluid glacial acetic acid
Formalin with which destroys
"post-chroming mitochondria and
" Regaud 's Golgi bodies of the
fluid (Muller 's cytoplasm. They
fluid) Orth 's have a pH of more
fluid than 4.6

Histochemical are those that

Fixatives preserve the
chemical
Formal Saline constituents of
10% Absolute cells and tissues.
Ethyl Alcohol
Acetone
Newcomer's
Fluid
Decalcifying Description Mechanism Advantages Disadvantage Specific
agent of Action Use

Chelating combine with The tissue is 1. It permits 1. It is very decalcification

agents calcium ions usually placed excellent slow, and is in EDTA using
and other salts in EDTA from staining therefore not a microwave
(e.g. iron and 1-3 weeks for results. recommended oven, addition
magnesium small 2. It produces for urgent and of ammonium
deposits) to specimens, but minimal cell routine hydroxide to
form weakly it may take 6-8 and tissue purposes. the EDTA
dissociated weeks or distortion. 2. It causes solution,
complexes and longer to 3. It forms slight tissue electrolytic
facilitate totally minimal hardening. decalcification,
removal of decalcify histological 3. EDTA or a one-step
calcium salt. dense cortical artifacts, inactivates fixation–decal
The most bone. The usually caused alkaline cification in
common solution should by production phosphatase formalin
chelating agent be changed of CO2 activity, which mercuric
in the market every 3 days, bubbles. can be restored chloric acid
is ethylene and in the final by addition of solution,
diamine tetra stage, every magnesium which also
acetic acid day, to chloride. appears
(EDTA) salt facilitate suitable for
decalcification mRNA In Situ
Hybridization,
might reduce
the time of
decalcification
considerably.

Ion Ion exchange A layer of the 1. Cellular The degree of

Exchange resin ion exchange detail is decalcification
Resin (ammonium resin, about well-preserved cannot be
form of 1/2 inch thick . 2. Daily measured by
polystrene is spread over washing of chemical
resin) hastens the bottom of solutions is means. 2. It is
decalcification the container eliminated. very slow, and
by removing to be used and 3. It permits is therefore not
calcium ions the specimen excellent recommended
from formic is placed on staining for urgent and
acid-containin top of it. The results. routine
g decalcifying decalcifying purposes.
solutions, agent is then
thereby added, usually
increasing 20-30 times
solubility from the volume of
the tissue. It is the tissue. The
not tissue may be
 recommended allowed to stay
for fluids in solution for
containing 1-14 days. The
mineral acids degree of
such as nitric decalcification
acid or may then be
hydrochloric measured by
acid physical or
X-ray method.
The resin that
has been
previously
used may later
be reactivated
by immersing
it in N/10
HCltwice and
washing it
with distilled
water three
times.

Electrophoresi Electrophoresi This method is Good

s s is a process satisfactory for cytologic and
(Electrical whereby small bone histologic
Ionization) positively fragments, details are,
charged processing however, not
calcium ions only a limited always
are attracted to number of preserved in
a negative specimens at a tissues that
electrode and time. have been
subsequently electrically
removed from decalcified.
the
decalcifying
solution.

Microwave The Microwave The quality of

oven microwave decalcification microwave-fix
decalcification oven has been is a novel ed tissues, at
used quite technique the respective
often for tissue compared to optimal time
processing, but the manual points, is
there are very method. In this comparable
few studies method, hard with routinely
describing its tissues are fixed tissues.
use in placed in the With the
decalcification decalcifying ability to have
of bone or agent in a entirely fixed
teeth. microwave tissues 3 hours
Microwave oven for after autopsy,
oven intermittent tissues can be
decalcification periods with harvested in
is faster than regular the morning,
routine changes of the placed on the
decalcification solution till the processor in
irrespective of end point is the afternoon,
the reached. and embedded
decalcifying Microwave the following
agents used. irradiation has morning.
been shown to
speed up the The uniformity
process of of the section’s
decalcification thickness is
significantly–f always
rom days to desirable
hours.