FIXATION AND FIXATIVES

MARK LESTER B. CAUAN, RMT

Histotechnology

 The art and science performed by the

histotechnologist to produce a tissue section
of good quality that will enable the
pathologist to diagnose the presence or
absence of disease.
 The first and most critical step in

histotechnology involves fixing or preserving

fresh tissue for examination. The process is
known as fixation.
FIXATION

 No process of histotechnology is more critical

to slide preparation than fixation.
 If the fixation is not adequate, the other

processes will also be inadequate.

 Inadequate or poor fixation will result in a

poorly processed tissue and will make it

difficult for the pathologist to render a proper
diagnosis.
 The primary aim of fixation is to preserve the

morphologic and chemical integrity of the cell

FIXATION

 With fixation, the shape, structure, intercellular

relationship and chemical constituents of tissues are
preserved.
 Fixation prevents degeneration, decomposition,

putrefaction, and distortion of tissues after removal

from the body.
 The secondary goal of fixation is to harden and

protect the tissue from the trauma of further

handling, so that it is easier to cut during gross
examination.
 Neutral buffered formalin allows a diagnosis to be

made in most cases.

FIXATION

 However, certain specialized studies such as

electron microscopy, histochemistry, and
immunocytochemistry require specific
fixation procedures.
 The most important reactions for maintaining

morphology in the fixation of tissues for

routine histopathology are those that
stabilize the proteins.
 Fixatives have the property of forming cross-

links between proteins.

FIXATION

To preserve the tissue

 Fixation preserves the tissue by stopping all

cellular activities so that the cells can be

viewed under the microscope as if they are
still in their original living state.
FIXATION

To prevent breakdown of cellular elements

 Surgical removal of the tissue from the body

will deprive it of oxygen and nutrition,

leading to a degradative chemical process or
cell death.
 Lysosome contain hydrolytic enzymes that

are released when the integrity of the cell is

destroyed.
FIXATION

To prevent breakdown of cellular elements

 Fixative prevents autolysis by inactivating the

lysosomal enzymes, or by chemical altering,

stabilizing and making the tissue
components insoluble.
 Fixation also protects the tissue from further

decomposition (putrefaction) after death due

to bacterial or fungal colonization and
overgrowth.
FIXATION

 To coagulate or precipitate Protoplasmic

substances
 Fixation renders insoluble certain tissue

component that may otherwise leak out

subsequent histologic handling.

 Two basic mechanisms involved in fixation:

 Additive fixation
 Non-additive fixation
Two basic mechanisms involved in fixation:

Additive fixation
 Whereby the chemical constituent of the fixative is
taken in and becomes part of the tissue by forming
cross-links or molecular complexes and giving
stability to the protein.
 Formalin, mercury, and osmium tetroxide)
Non-additive fixation
 The fixing agent is not incorporated into the tissue,
but alters the tissue composition and stabilizes the
tissue by removing the bound water attached to H
bonds of certain groups within the protein molecule
 Alcoholic fixatives.
MAIN FACTORS INVOLVED IN FIXATION

Hydrogen ion Concentration

 Satisfactory fixation occurs between ph 6 to 8.
 Outside this range, changes may occur that are

detrimental to ultrastructural preservation of the tissue.

Temperature
 Room temperature

 For EM, and some histochemistry , 0-4C

 Formalin heated to 60C is sometimes used for the rapid

fixation of very urgent biopsy, although the risk of

tissue distortion is increased.
 Formalin at 100C can be used to fix tissues with

tuberculosis.
MAIN FACTORS INVOLVED IN FIXATION

Thickness of the section

 Tissue blocks taken should be either small (1-2

mm for EM and 2cm for light microscopy) or thin

(no more than 0.4cm for light microscopy) or as
prescribed by tissue processor manufacturer
 Large specimen, should be opened or sliced

thinly.
 Brain should be suspended whole in 10%buffered

formalin for 2-3 weeks to ensure fixation and

some hardening prior to sectioning.
MAIN FACTORS INVOLVED IN FIXATION

Osmolality
 Hypertonic solutions give rise to cell

shrinkage.
 Isotonic as well as hypotonic fixatives cause

cell swelling and poor fixation.

 The best results are usually obtained using

slightly hypertonic solutions ( 400-

450mOsM), isotonic solutions (340mosm)
MAIN FACTORS INVOLVED IN FIXATION

Concentration
 Formaldehyde is normally used as a 10%

solution and glutaraldehyde is normally used

as 3% solutions
 The presence of buffer causes polymerization

of the aldehyde, with consequent decrease in

its effective concentration.
 
MAIN FACTORS INVOLVED IN FIXATION

Duration of fixation
 Primary fixation in buffered formalin is usually

carried out for 2-6 hours during the day the

specimen is obtained, but they remain in fixatives
over the week end without much adverse effect.
 Prolonged fixation may cause shrinkage and

hardening of tissue, and may severely inhibit

enzyme activity and immunological reactions.
 For EM, it is recommended that diced tissues be

fixed for 3 hours and then placed in holding

buffer.
Practical consideration of Fixation

Speed
 Specimen should be placed in fixatives as

soon as it is removed from the body. This is

done to prevent autolysis and putrefaction
Penetration
 1mm/hr
 Slows down as it goes deeper into the tissue
Practical consideration of Fixation

Volume
 10-25 times the volume of tissue to be fixed
 Maximum effectiveness of fixation is noted to

be 20 times the tissue volume

Duration of Fixation
 Fibrous organs such as uterus or intestinal

tract take longer than small or loosely textured

tissues such as biopsies or scrapings.
 Fixation time can be cut down by using heat,

vacuum, agitation or microwave.

Miscellaneous consideration
 The tissue selected for sectioning should be
thin enough to allow penetration by fixatives
within reasonable amount of time.
 To maintain an adequate fixation time of 4-

6hours, the recommended size of the tissues

is 2cm square, and no more than 4mm thick.
 Most tissues can be cut and trimmed without

prior fixation, except for the brain which is

generally soft when unfixed.
 Refrigeration is used to slow down

decomposition if the tissue needs to be

photographed and cannot be fixed
immediately.
Effects of Fixatives in general

 They harden soft and friable tissues and

make the handling and cutting of sections
easier. Accelerated by the action of alcohol
during the dehydration process.
 They make the cells resistant to damage and

distortion caused by hypotonic and

hypertonic solutions used during tissue
processing.
 Inhibit bacterial decomposition
Effects of Fixatives in general

 Increase optical differentiation of cells and

tissue components thereby rendering them
more readily visible during examination.
 Act as a mordant or accentuators to promote

or hasten staining or they may inhibit certain

dyes in favor of another. (formaldehyde
intensifies while osmium tetroxide inhibits
hematoxylin staining)
 Reduce the risk of infections during handling

and actual processing of tissues.

Characteristics of a Good Fixatives

 Cheap
 Stable
 Safe to handle
 Kill the cell quickly thereby producing

minimum distortion of cell constituent

 Inhibit bacterial decomposition and autolysis
 Produce minimum shrinkage of tissue
Characteristics of a Good Fixatives

 Harden tissues thereby making the cutting of

sections easier.
 Must be isotonic
 Must make cellular components insoluble to

hypotonic solutions and render them

insensitive to subsequent processing
 Permit subsequent application of many

staining procedures to facilitate easier and

profitable examination.
 Sofar, no single fixatives has yet
been known to possess all the
qualities of an ideal solution.
There are numerous fixatives
available, and each has its own
advantages and disadvantages.
 TYPES OF FIXATIVES
ACCORDING TO ACTION AND
COMPOSITION
According to COMPOSITION

 Simple fixatives
◦ Aldehydes
 Formaldehyde
 glutaraldehyde
◦ Metallic fixatives
 Mercuric chloride
 Chromate fixatives
 Potassium dichromate
 Chromic acid
 Lead fixatives
 Picric acid
 Acetic acid
 Acetone
 Alcohol
 Osmium teroxide (osmic acid)
 heat
According to COMPOSITION
 Compound fixatives

◦ made up of two or more fixatives which have been

added together to obtain the optimal combined
effect of their individual actions upon the cells and
tissue constituent.
According to ACTION:

 Microanatomical fixatives
◦ Those that permit the general microscopic study of
tissue structures without altering the structural pattern
and normal intercellular relationship of the tissues in
question.

 Cytological
◦ Those that preserve specific parts and particular
microscopic elements of the cell itself
◦ Nuclear fixatives
◦ Cytoplasmic fixatives
◦ Histochemical Fixatives
Microanatomical fixatives

 10% fomol saline

 10% buffered neutral formalin
 Heidenhains Susa
 Formol sublimate (formol corrosive)
 Zenker’s solution
 Zenker-formol (kelly’s solution)
 Bouin’s solution
 Brasil solution
Cytological fixatives
◦ Nuclear fixatives
 Preserves the nuclear structures
 Contain glacial acetic acid as their primary
component due to its affinity for nuclear
chromatin.
 Ph of 4.6 or less
 Flemming’s fluid
 Carnoy’s fluid
 Bouin’s fluid
 Newcomer’s fluid
 Heidenhain’s Susa
Cytological fixatives
Nuclear fixatives
 Ethanol and methanol, including Carnoy’s
solution, are commonly used fixatives for
nucleic acids.
 Ethanol appears to give the most valuable
usable DNA fragments for PCR, whereas
formaldehyde limits the size of fragment s
which can be retrieved.
Cytological fixatives
◦ Cytoplasmic fixatives
 Preserves cytoplasmic structures
 Must never contain glacial acetic acid which destroys
mitochondria and golgi bodies of the cytoplasm.
 ph is more than 4.6
 Flemming’s fluid without acetic acid
 Kelly’s fluid
 Formalin with “post-chroming”
 Regaud’s fluid (Muller fluid)
 Orth’s fluid
 For RNA, the precipitant fixatives ethanol and
acetone give the best quantitative results using
frozen tissues as the standard.
Cytological fixatives
◦ Histochemical Fixatives
 Preserves the chemical constituents of cells and
tissues
 Formol saline 10%
 Absolute Ethyl alcohol
 Acetone
 Newcomer’s fluid
LIPID FIXATION

 Cryostat or frozen sections should be used

for demonstrating lipid in tissues, followed by
a general lipid stain.
 Fixatives containing mercuric chloride and

potassium dichromate can be effective for

preservation of lipids in cryostat sections.
 In general, phospholipids, which contain

amino groups, are fixed by aldehydes.

 Baker’s formol-calcium may be used to

preserve phospholipids.
CARBOHYDRATE FIXATION
 Alcoholic fixatives are generally
recommended for glycogen fixation.
Glycogen can be demonstrated satisfactorily
enough for diagnosis, although losses of
glycogen can be high (60-80%) in aqueous
solution.
 Alcoholic formaldehyde is a better fixative in

human skin compared with neutral buffered

formaldehyde.
PROTEIN FIXATION

 Neutral buffered formol saline or

formaldehyde vapor are the most commonly
used fixatives for amino acids.
GLYCOGEN FIXATION

 Most useful fixatives are alcohol based, such

as Rossman’s fluid or cold absolute alcohol.
 Essential when processing tissues from

patients with glycogen storage disease.

 There is better retention of glycogen if the

section is coated with celloidin.

MIXTURES OF FIXATIVES

 Two aldehyde fixative mixtures have been

particularly useful for Electron cytchemistry.
 The best known is Karnovsky’s
paraformaldehyde-glutaraldehyde solution.
 Acrolein is another aldehyde which has been
introduced as a mixture with glutaraldehyde or
formaldehyde.
 Acrolein penetrates tissues rapidly, preserves
morphology and enzyme activity at low
concentrations, and may be useful for immersion
fixation of surgical biopsies.
ALDEHYDE FIXATIVES
 Formaldehyde (Formalin)
 10% Formol-Saline
 10% Neutral buffered formalin or phosphate –

Buffered formalin
 Formol-Corrosive (Formol Sublimate)
 Alcoholic Formalin (Gendre’s) Fixative
 GLUTARALDEHYDE
Formaldehyde (Formalin)
 Most widely used fixatives is 10% formalin.
 A gas produced by the oxidation of methyl

alcohol and is soluble in water to the extent of

37-40% weight in volume.
 The commercially available solution of

formaldehyde contains 35-40% gas by weight.

 Pure stock solution of 40 % formalin is

unsatisfactory for routine fixation since high

formaldehyde concentrates tend to over harden
the outer layer of the tissue and affect staining
adversely
Formaldehyde (Formalin)
Formaldehyde (Formalin)
 Commonly used as 4% solution, giving 10%
formalin for tissue fixation.
 It must be diluted 1:10 to make 10 %

solution.
 Usual fixation time is 24 hours
 buffered to pH 7 with phosphate buffer
Formaldehyde (Formalin)
Advantages
 cheap, readily available, easy to prepare, and relatively

stable, especially if stored in buffered solutions.

 Compatible with many stains
 Does not over harden tissues, even with prolonged

periods of fixation, as long as solutions are regularly

changed.
 Penetrates tissue well.
 Preserves fat and mucin, making them resistant to

subsequent treatment with fat solvents, thereby

allowing them to be stained for demonstration.
 Preserves glycogen
Formaldehyde (Formalin)
 Advantages
 It preserves but not precipitate proteins, thereby

allowing tissue enzymes to be stained.

 Recommended for nervous tissue preservation.
 Allows natural tissue color to be restored after fixation

by immersing formalin fixed tissues in 70% alcohol for

one hour. Recommended for colored tissue photography.
 Allows frozen tissue sections to be prepared easily.
 Does not require washing out, unless tissues have stayed

in formalin for excessively long periods of time.

 It is a tolerant fixative, used for mailing specimen

because specimen can be left in formalin indefinitely.

Formaldehyde (Formalin)
 Disadvantages
 Fumes are irritating to the nose and eyes and

may cause sinusitis, allergic rhinitis or

excessive lacrimation
 Irritating to the skin and may cause allergic

dermatitis.
 Produce considerable shrinkage of tissues
 Soft fixative and does not harden some

cytoplasmic structures adequately enough for

paraffin embedding
Formaldehyde (Formalin)
 Disadvantages
 If unbuffered:

◦ Reduces both basophilic and eosinophilic staining of

cells, thereby reducing the quality of routine cytologic
staining.
◦ Forms abundant brown pigment granules on blood-
containing tissues
 Prolonged fixation may produce:
◦ Bleaching of the specimen and loss of natural tissue
colors
◦ Dispersal of fat from the tissues into the fluid
◦ Dissolution or loss of glycogen
PRECAUTIONS:

 Prolonged storage of formaldehyde, especially at

very low temperature, may induce precipitation
of white paraformaldehyde deposits and produce
turbidity although this, in itself, does not impair
the fixing property of the solution.
 Methanol added as a preservatives to
formaldehyde will prevent its decomposition to
formic acid or precipitation to
paraformaldehyde, but it serves to denature
protein, thereby rendering formalin unsuitable
as a fixative for EM.
PRECAUTIONS:

 Concentrated solutions of Formaldehyde must

NEVER be neutralized since this might precipitate
violent explosions.
 Room should be properly ventilated with adequate
windows and preferably with exhaust fan to
prevent inhalation of fumes and consequently
injury to the eyes and nose.
 Dermatitis may be avoided by the use of rubber
gloves when handling specimens fixed in formalin.
 Bleaching of tissues may be prevented by
changing the fluid fixative every three months.
PRECAUTIONS:

 Natural tissue colors may be restored by immersing

tissues in 70% alcohol after fixation.
 Brown or black crystalline precipitate formed by the
cation of formic acid with blood can be removed
from the sections prior to staining by treatment with
saturated alcoholic picric acid or a 1% solution of
potassium hydroxide in 80% alcohol. The use of
neutral buffered formalin will prevent the
pigmentation.
 If fatty tissues are to be stored for a long time,
cadmium or cobalt salts are added to prevent
dispersion of fat out into the fluid.
PRECAUTIONS:

 Acid reaction due to formic acid formation can

be buffered or neutralized by adding
magnesium carbonate or calcium carbonate to
10-15% formalin. This should be done on a
wide mouth bottle to prevent violent explosion
due to insufficient gas space for CO2 release.
 To improve staining and produce firmer and

harder consistency, tissues fixed in formalin for

1-2 hours may be placed again in helly’s fluid
for 4-6 hours or in formol-sublimate for 4-16
hours (secondary fixation)
PRECAUTIONS:

 If postfixed in osmic acid, the tissue must not be

washed in demineralized water to prevent
hypotonicity and bleaching.
 Fixation of tissue blocks not exceeding 5mm in

thickness is usually complete in 6-12 hours at

room temperature.
 Fixation by formalin is influenced by heat, vacuum,

agitation and microwave techniques. Fixation can

be speeded up by heating solution at 50C and
placing it under a vacuum or in microwave.
  
10% Formol-Saline

 Simple microanatomical fixative made up of saturated

formaldehyde (40% weight volume) diluted to 10% with
sodium chloride.
 Recommended for fixation of CNS tissues and general

post-mortem tissues for histochemical examination.

 Formula

◦ Formaldehyde, 40%-----------100ml
◦ NaCL ___________________________9grams
◦ Distilled water __________________900 ml
 Fixation time ----24 hours at 35 C (95F)
 48 hours at 20-25 C (65-77 F)
  
10% Formol-Saline
 Advantages:

 Penetrates and fixes tissues evenly

 Preserves microanatomic and cytologic details

with minimum shrinkage and distortion

 Large specimens may be fixed for long time

provided that the solution is changed every

three months
 Preserves enzymes and nucleoproteins
10% Formol-Saline
 Advantages:

 Demonstrates fats and mucin

 Does not over harden tissues, thereby

facilitating dissection of the specimen

 Ideal for most staining techniques, including

silver impregnation
 Allows natural tissue color to be restored

upon immersion in 70%alcohol.

10% Formol-Saline
Disadvantages

 Slow fixative
 Tissues tend to shrink during alcohol

dehydration, this may reduced secondary

fixation
 Metachromatic reaction of amyloid is reduced
 Acid dye stains less briefly than when fixed

with mercuric chloride.

10% Neutral buffered formalin or phosphate –
Buffered formalin
 Recommended for preservation and
storage of surgical, postmortem and
research specimens.
 Formula
◦ Sodium dihydrogen phosphate (anhydrous) –3.5 grams
◦ Disodium hydrogen phosphate – 6.5 grams
◦ Formaldehyde 40% 100ml
◦ Distilled water 900ml
 Fixation time - 4-24 hours
10% Neutral buffered formalin or phosphate –Buffered
formalin

 Advantages
◦ Similar to formol saline
with the following additions
◦ Prevents precipitation of
acid formalin pigments on
postmortem tissues
◦ Best fixatives for tissues
containing iron pigments
and for elastic fibers
◦ Requires no post treatment
after fixation goes directly
into 80% alcohol processing
10% Neutral buffered formalin or phosphate –
Buffered formalin
 Disadvantages
◦ Longer to prepare, time consuming
◦ Positivity of mucin to PAS is reduced
◦ May produce gradual loss in basophilic staining
of cells
◦ Reactivity of myelin to Weigert’s iron hematoxylin
stain is reduced
◦ Inert towards lipids
Formol-Corrosive (Formol Sublimate)

 Formol mercuric chloride solution is

recommended for routine post mortem tissue

 Fixation time – 3-24 hours

Formol-Corrosive (Formol Sublimate)

 Advantages
◦ Penetrates small pieces of tissues rapidly
◦ Produces minimum shrinkage and hardening
◦ Excellent for many staining procedures including
silver reticulum methods
◦ Brighten cytoplasmic and metachromatic stains
better than with formalin alone.
◦ Cytological structures and blood cells are well
preserved
◦ No need for washing out
◦ Fixes lipid
Formol-Corrosive (Formol Sublimate)

 Disadvantages
◦ Penetration is slow, sections should not be more
than 1cm thick
◦ Forms mercuric chloride deposits
◦ Does not allow frozen section
◦ Inhibits the determination of the extent of tissue
decalcification.
Alcoholic Formalin (Gendre’s) Fixative

 Formula
◦ 95% ethyl alcohol saturated with picric acid 80ml
◦ Strong formaldehyde solution 15ml
◦ Glacial acetic acid 5ml
 Postfixation with phenol-formalin for 6
hours or more can enhance
immunoperoxidase studies on the tissues,
and in some cases, EM, if it is necessary at a
later time to establish a diagnosis
Alcoholic Formalin (Gendre’s) Fixative

 Advantages
◦ Fixation is faster
◦ Used for rapid diagnosis
◦ Good for the preservation of glycogen and for
micro-incineration technique
◦ Used to fixed sputum
Alcoholic Formalin (Gendre’s) Fixative

 Disadvantages
◦ Produce gross hardening of tissues
◦ Causes partial lysis of RBC
◦ Preservation of iron containing pigments is poor
◦ Formaldehyde does not give as good a
morphological picture as glutaraldehyde
◦ Causes little cross-linking
GLUTARALDEHYDE

 Made up to two formaldehyde residues,

linked by three carbon chains
 Buffered glutaraldehyde, followed by

secondary fixation in osmium tetroxide is

satisfactory for EM
 Fixation time varies from half hour to two

hours.
GLUTARALDEHYDE

 Advantages
◦ It has more stable effect on tissues, giving a
firmer texture with better tissue sections,
especially of CNS.
◦ Preserves plasma proteins better
◦ Produces less tissue shrinkage
◦ Preserves cellular structures better,
recommended for enzyme histochemistry and EM
◦ More pleasant and less irritating to the nose
◦ Does not cause dermatitis
GLUTARALDEHYDE

 Disadvantages
◦ More expensive
◦ Less stable
◦ Penetrates tissues more slowly
◦ Tends to make tissue brittle
◦ Reduces PAS positivity of reactive mucin
GLUTARALDEHYDE

 Precaution:
◦ Specimen vial must be kept refrigerated during
the fixation process
◦ Solution may be changed several times during
fixation by swirling the vials to make sure that
the specimen is in contact with fresh solution all
the time.
METALLIC FIXATIVES

 MERCURIC CHLORIDE
◦ Zenker’s fluid
◦ Zenker-formol (helley’s solution)
◦ Heidenhain’s Susa solution
◦ B5 fixative

 CHROMATE FIXATIVES
◦ Chromic acid
◦ Potassium dichromate
◦ Regaud’s (Muller) Fluid
◦ Orth’s fluid

 LEAD FIXATIVES
MERCURIC CHLORIDE

 Mercuric chloride is the most common metallic

fixative, frequently used in saturated aqueous
solutions of 5-7%
 Widely used as secondary fixative reacting with a
number of amino acid residues and accompanied by
spectroscopic changes, probably due to reaction
with histidine residues.
 Penetrates poorly and produces shrinkage of tissues
 Tissues fixed with mixtures containing mercuric
chloride (except susa) contain black precipitates of
mercury.
Mercuric chloride
MERCURIC CHLORIDE

 Advantages
◦ Penetrates and hardens tissues rapidly and well
◦ Nuclear components are shown in fine detail
◦ Precipitates all protein
◦ Greater affinity to acid dyes
◦ Trichrome staining is excellent
◦ Routine fixative of choice for preservation of cell
detail in tissue photography
◦ Permits brilliant metachromatic staining of cells
◦ Recommended for renal tissues, fibrin, connective
tissues and muscles
MERCURIC CHLORIDE

 Disadvantages
◦ Causes marked shrinkage of cells
◦ Rapidly hardens the outer layer of the tissue with
incomplete fixation of the center
◦ Penetration beyond the first 2-3 mm is` slow,
should not used more than 5mm
◦ If left in fixative for more than 1-2 days, the
tissue becomes unduly hard and brittle
◦ Prevents adequate freezing of fatty tissues and
makes cutting of frozen tissues difficult
MERCURIC CHLORIDE

 Disadvantages
◦ Causes lysis of RBC and removes iron from
hemosiderin
◦ Inert to fats and lipids
◦ Leads to formation of black granular deposits in
the tissues.
◦ Reduces the amount of demonstrable glycogen
◦ Compound solutions containing mercuric chloride
deteriorate rapidly upon addition of glacial acetic
acid to formalin
◦ Extremely corrosive to metals
MERCURIC CHLORIDE

 Precautions
◦ Black deposits may be removed by adding saturated
iodine solution in 96% alcohol, the iodine being
decolorized with absolute alcohol in the subsequent
dehydration .
◦ Compound solutions must always be freshly
prepared.
◦ The use of metallic forceps and of metal caps to
cover the bottles containing the fixative should be
avoided.
◦ Contact of mercuric fixatives with personal
jewelries should be avoided
MERCURIC CHLORIDE

 Mercuric chloride stock solution formula

◦ Mercuric chloride 5grams
◦ potass ium dichromate 2.5grams
◦ sodium sulfate 1 gram
◦ Distilled water 100ml
 Just before use, glacial acetic acid may be
added to form Zenker’s solution or strong
formaldehyde solution may be added to
form Zenker-Formol solution (Kelly’s)
Zenker’s fluid

◦ Made up of mercuric chloride stock solution to

which glacial acetic acid has been added just
before its use to prevent turbidity and formation
of a dark precipitate.
◦ Recommended for fixing small pieces of liver,
spleen, connective tissue fibers and nuclei.
◦ Fixation time – 12-24hours
◦ Formula
 Mercuric stock solution 95ml
 Glacial acetic acid 5ml
Zenker’s fluid

◦ Advantages
 Produces a fairly rapid and even fixation of
tissues
 Stock solution keep well without disintegration
 Recommended for trichrome staining
 Permits brilliant staining of nuclear and
connective tissue fibers
 Compatible with most stains
 May act as a mordant to make certain special
staining reactions possible
Zenker’s fluid

◦ Disadvantages
 Penetration is poor
 Not stable after addition of Acetic acid
 Prolonged fixation will make tissues brittle and hard
 It causes lysis of red blood cells and removes iron from
hemosiderin
 Does not permit cutting of frozen sections
 Tendency to form mercuric pigment deposits or
precipitates
 Tissue must be washed in running water for several
hours before processing. Insufficient washing may
inhibit or interfere with good cellular staining
Zenker’s fluid
◦ Precautions and practical considerations
 Do not left tissues stay in solution for more than
24 hours
 Solutions must always be freshly prepared
 Tissues must be washed out thoroughly in running
water to permit good staining.
 Mercuric deposits may be removed by immersing
tissues in alcoholic iodine solution prior to staining,
through a process known as de-zenkerization
 Tissues should be cut thin (2-3mm)and hollow
organs should be opened to promote complete
penetration and fixation.
Zenker-formol (helley’s solution)

 Fixation time 12-24 hours

Zenker-formol (helley’s solution)

◦ Advantages
 Excellent microanatomic fixative for pituitary
gland, bone marrow and blood containing
organs such as spleen and liver
 Penetrates and fixes tissues well
 Nuclear fixation and staining is better than
zenker’s
 Preserves cytoplasmic granules well
Zenker-formol (helley’s solution)

◦ Disadvantages
 Similar with Zenker’s except that brown
pigments are produced if tissues are allowed to
stay in the fixative for more than 24 hours due
to RBC lysis.
 This may be removed by immersing the tissue
in saturated alcoholic picric acid or sodium
hydroxide
Heidenhain’s Susa solution

◦ Recommended for skin biopsies

◦ Excellent cytologic fixative
◦ Formula
 Mercuric chloride 45grams
 Sodium chloride 5grams
 Trichloroacetic acid 20grams
 Glacial acetic 40ml
 Formaldehyde, 40 % 200ml
 Distilled water 800ml
Fixation time 3-12 hours
Heidenhain’s Susa solution

◦ Advantages
 Penetrates and fixes tissues rapidly and evenly
 Produces minimum shrinkage and hardening of tissues
due to counter-balance of the swelling effects of acids
and the shrinkage effect of mercury.
 Permits most staining procedures to be done, including
silver impregnation, producing brilliant results with
sharp nuclear and cytoplasmic details
 Permits easier sectioning of large blocks of fibrous
connective tissues
 Susa-fixed tissues may be transferred directly to 95%
alcohol or absolute alcohol, thereby reducing
processing time.
Heidenhain’s Susa solution

◦ Disadvantages
 Prolonged fixation of thick material may
produce considerable shrinkage, hardening and
bleaching.
 RBC preservation is poor
 Some cytoplasmic granules are dissolved
 Mercuric chloride deposits tend to form on
tissues
 Weigert’s method of staining elastic fibers is not
possible in susa-fixed tissues
Heidenhain’s Susa solution

◦ Precaution
 After using Heidenhain’s Susa fixative, the
tissue should be transferred directly to a high
grade alcohol, to avoid undue swelling of
tissues caused by treatment with low grade
alcohol or water.
B5 fixative

◦ Commonly used for Bone marrow biopsies

◦ Formula
 Distilled water 90ml
 Mercuric chloride 6grams
 Sodium acetate 1.25 grams

◦ Just prior to use, add 1cc of formaldehyde (40%)

for 10 cc of B5.
B5 fixative

◦ Advantages

 Rapid fixation can be achieved in ½ to 2 hours

 Good fixative for cytology of bone marrow
biopsies
B5 fixative

◦ Precautions and practical considerations

 Overfixation hardens the tissue and make
cutting difficult
 Two working solutions are kept separate , since
the mixture is unstable. Mix just prior to use.
 Mercury pigments can be removed by de-
zerkerization.
CHROMATE FIXATIVES

 Chromic acid
 Potassium dichromate
 Regaud’s (Muller) Fluid
Chromic acid

◦ Used in 1-2 % aqueous solution, usually as a

constituent of a compound fixative
◦ Precipitates all proteins and adequately preserves
carbohydrates
◦ Strong oxidizing agent, hence a strong reducing
agent must be added to chrome containing
fixatives before use in order to prevent
counteracting effects and consequent
decomposition of solution upon prolonged
standing.
Potassium dichromate

◦ Used in 3% aqueous solution

◦ Fixes but not precipitate cytoplasmic structures
◦ Preserves lipids
◦ Preserves mitochondria(if used in pH 4.5-5.2)
Regaud’s (Muller) Fluid

◦ Formula
 Potassium dichromate 3%----------------------80ml
 Strong formaldehyde 40%----------------------20ml
 To be added just before use
◦ Fixation time -------12-48hours
Regaud’s (Muller) Fluid

◦ Advantages
 Penetrate tissues well
 Harden tissues better and more rapidly than
orth’s fluid
 Recommended for demonstration of chromatin,
mitochondria, mitotic figures, Golgi bodies, RBC
and colloid containing tissues.
Regaud’s (Muller) Fluid

◦ Disadvantages
 Deteriorates and darkens on standing due to
acidity, hence the must always be freshly
prepared.
 Penetration is slow
 Chromate fixed tissues tend to produce
precipitates of sub oxide, hence should be
thoroughly washed in running water prior to
dehydration.
Regaud’s (Muller) Fluid

◦ Disadvantages
 Prolonged fixation blackens tissue pigments
 Glycogen penetration is poor
 Does not preserves fats
 Preserves hemosiderin less than buffered
formation
 Intensity of PAS reaction is reduced
Orth’s fluid

◦ Formula
 Potassium dichromate 2.5% -------------------100ml
 Sodium sulfate (optional)--------------------------1gram
 Strong formaldehyde 40%-------------------------10ml
to be added just before use
◦ Fixation time -36 to 72 hours
Orth’s fluid

◦ Advantages
 Recommended for the study of early
degenerative processes and tissue necrosis
 Demonstrate rickettsiae and other bacteria
 Preserves myelin better that buffered formalin
◦ Disadvantages----------------------same as
regaud’s fluid
LEAD FIXATIVES

 Used in 4% aqueous solution of basic lead

acetate
 Advantages

◦ Recommended for acid mucopolysaccharides

◦ Fixes connective tissue mucin
 Disadvantages
◦ Takes up CO2 to form insoluble lead carbonate
especially on prolonged standing. This may be
removed by filtration or by adding acetic acid
drop to lower the ph and dissolve the residue.
PICRIC ACID FIXATIVES

 Bouin’s solution
 Brasil’s alcoholic Picroformol Fixative
Bouin’s solution
 Recommended for fixation of embryos and
pituitary biopsies
 Formula

◦ Saturated solution of picric acid 75 ml

◦ Strong formaldehyde 40% 25ml
◦ Glacial acetic acid 5ml
 Fixation time -------------6-24 hours
Bouin’s solution
Bouin’s solution

 Advantages
◦ Produces minimal distortion of micro-anatomial
structures and can be used for general and
special stains
◦ Excellent fixative for preserving soft and delicate
structures
◦ Penetrates rapidly and evenly, causes little
shrinkage
 Yellow stain is useful when handling
fragmentary biopsies
Bouin’s solution

 Advantages
◦ Permits brilliant staining of tissues
◦ Preferred fixative for tissues to be stained by
Masson’s trichrome for collagen, elastic or
connective tissue
◦ Preserves glycogen
◦ Does not need “washing out”
Bouin’s solution

 Disadvantages
◦ Penetrates large tissues poorly, hence, its use is limited to
small fragments of tissue
◦ Picrates are soluble in water, hence, tissues should not be
washed in running water but rather, transferred directly
from fixative to 70% alcohol
◦ Not suitable for fixing kidney structures, lipid and mucus.
◦ Destroys cytoplasmic structures, mitochondria
◦ Produces RBC hemolysis and removes demonstrable ferric
iron from blood pigments.
◦ Reduces or abolishes Feulgen reaction due to hydrolysis of
nucleoproteins.
  
Brasil’s alcoholic Picroformol Fixative

 Formula
◦ Formaldehyde 37%-----------------------------2040ml
◦ Picric acid --------------------------------------80grm
◦ Ethanol or isopropyl alcohol ---------------------6000ml
◦ Trichloroacetic acid -----------------------------65grm
Brasil’s solution

 Advantages
◦ Better and less messy than bouin’s solution
◦ Excellent fixative for glycogen
 Overnight tissue fixation by automatic
processing technique may utilize 3-4
changes of Brasil’s fixative at ½ to 2 hours
each, succeeded directly by absolute
alcohol.
GLACIAL ACETIC ACID

 Acetic acid is normally used in conjunction

with other fixatives to form a compound
solution. Solidifies at 17C, hence, the name
glacial acetic acid.
 Advantages

◦ Fixes and precipitates nucleoproteins

◦ Precipitates chromosomes and chromatin
materials, hence, is very useful in the study of
nuclear components of the cell.
◦ Causes tissues to swell, to counteract the
shrinkage produced by other products
 Disadvantages
◦ When combine with Potassium Dichromate,
the lipid-fixing property of the later is
destroyed (zenker’s fluid).
◦ Contraindicated for cytoplasmic fixation
since it destroys mitochondria and golgi
elements of the cells.

GLACIAL ACETIC ACID

ALCOHOLIC FIXATIVES

 Methyl alcohol 100%

 Isopropyl Alcohol 95%
 Ethyl alcohol
 Carnoy’s fluid
 Newcomer’s Fluid
ALCOHOLIC FIXATIVES
 Alcohol rapidly denatures and precipitates proteins
by destroying hydrogen and other bonds.
 It must be used in concentrations ranging from 70
to 100% because less concentrated solutions will
produce lysis of cells.
 Absolute alcohol can be used to fix and preserve
glycogen, pigments, blood, tissue films and smears.
 The color of the specimen can be preserved for
photographic work using 80% alcohol. Glycerin is
also used in combination with alcohol for this
purpose.
ALCOHOLIC FIXATIVES
 Advantages
◦ Ideal for small tissue fragment
◦ Used both as fixative and dehydrating agent
◦ Excellent for glycogen preservation
◦ Preserves nuclear stain
ALCOHOLIC FIXATIVES
 Disadvantages
◦ Lower concentration (70-80%) will cause RBC
hemolysis and inadequately preserve leukocytes.
◦ Dissolves fats and lipids, as a general rule. ALCOHOL
CONTAINING FIXATIVES ARE CONTRAINDICATED
WHEN LIPIDS ARE TO BE STUDIED.
◦ Causes glycogen granules to move towards the poles
or ends of the cells (polarization)
◦ Tissue left in alcohol too long will shrink, making it
difficult or impossible to cut.
  
Methyl alcohol 100%

Advantages
 Excellent for fixing dry and wet smears, blood
smears and bone marrow tissues.
 Fixes and dehydrates at the same time.
Disadvantages
 Penetration is slow
 If left in fixative for more than 48hours, tissues may
be overhardened and difficult to cut
Isopropyl Alcohol 95%

 Used for fixing touch preparation, although some

touch preparations are air dried and not fixed, for
certain special staining procedures such as wright
-Giemsa.
Ethyl alcohol

◦ Used at concentration of 70-100%.

◦ If lower concentrations are used, the RBC’s
become hemolysed and WBC’s are inadequately
preserved.

◦ Fixation time --------------18-24 hours

Ethyl alcohol
Ethyl alcohol

◦ Advantages
 Preserves but not fix glycogen
 Fixes blood, tissue films and smears
 Preserves nucleoproteins and nucleic acids,
hence used for histochemistry especially for
enzyme studies.
 Fixes tissue pigments fairly well.
Ethyl alcohol
◦ Disadvantages
 Causes polarization of glycogen granules
 Produces considerable hardening and shrinkage
of tissues.
 Hemosiderin preservations is less than in
buffered formaldehyde
 Strong reducing agent, hence, should not be
mixed with chromic acid, potassium dichromate
and osmium tetroxide which are strong
oxidizing agents.
Carnoy’s fluid
◦ Recommended for fixing chromosomes, lymph
glands and urgent biopsies.
◦ Rapid in action and may be used for urgent
biopsy specimens for paraffin processing within 5
hours.
◦ Tissues fixed with carnoy’s fixative for 1 hour can
be transferred directly to absolute alcohol or an
alcohol-chloroform mixture (1:1).
◦ It is also used to fix brain tissue for diagnosis of
rabies
Carnoy’s fluid
◦ Formula
 Absolute alcohol------------------60ml
 Chloroform----------------------30ml
 Glacial acetic acid ----------------10ml

◦ Fixation time 1-3 hours

Carnoy’s fluid
◦  Advantages
 Most rapid fixative
 Fixes and dehydrates at the same time
 Permits good nuclear staining and differentiation
 Preserves Nissl granules and cytoplasmic granules well
 Preserves nucleoproteins and nucleic acids
 Excellent fixative for glycogen since aqueous solutions
are avoided.
 Very suitable for small tissue fragments such as
curettings and biopsy materials
 Following fixation, tissue may be transferred directly to
absolute alcohol, thereby shortening processing time.
Carnoy’s fluid
◦ Disadvantages
 Produces RBC hemolysis
 Causes considerable tissue shrinkage
 Suitable only for small pieces of tissues due to
slow penetration.
 Tends to harden tissues excessively and distorts
tissue morphology.
 Dissolves fat, lipids, and myelin
 Leads to polarization unless very cold
temperature (-70C) are used
 Dissolves acid-soluble cell granules and pigments
Newcomer’s Fluid

◦ Formula
 Isopropyl alcohol ------------------60ml
 Propionic acid---------------------30ml
 Petroleum ether -------------------10ml
 Acetone--------------------------10ml
 Dioxane--------------------------10ml

 Fixation time -----------------12-18hours

Newcomer’s Fluid

◦ Advantages
 Recommended for fixing mucopolysaccharides
and nuclear proteins
 Produces better reaction in Feulgen stain than
Carnoy’s fluid
 Acts both as nuclear and histochemical fixative.
OSMIUM TETROXIDE (OSMIC ACID)

 Pale yellow powder which dissolves in water

(up to 6% at 20C) to form a strong oxidizing
solution.
 Causes complete denaturation of protein
 Potassium permanganate and potassium

dichromate are also oxidizing agents but are

less reactive towards proteins than in osmium
tetroxide
OSMIUM TETROXIDE (OSMIC ACID)

 Advantages
◦ Fixes conjugated fats and lipids permanently by
making them insoluble during subsequent
treatment with alcohol and xylene
◦ Preserves cytoplasmic structures well
◦ Fixes myelin and peripheral nerves
◦ Produces brilliant nuclear staining with safranin
OSMIUM TETROXIDE (OSMIC ACID)

 Advantages
◦ Adequately fixes material for ultrathin sectioning
in EM, since it rapidly fixes small pieces of tissues
and aids in their staining.
◦ Precipitates and gels proteins
◦ Shows uniformly granular nuclei with clear
cytoplasmic background
◦ Some tissues (adrenal gland) are better fixed in
vapor form of osmium tetroxide.
OSMIUM TETROXIDE (OSMIC ACID)

 Disadvantages
◦ very expensive
◦ poor penetrating agent, suitable only for small pieces of
tissues (2-3mm thick)
◦ readily reduced by contact with organic matter and
exposure to sunlight , forming a black precipitate which
settles at the bottom of the container.
◦ Prolonged exposure to acid vapor can irritate the eye,
producing conjunctivitis or cause the deposition of black
osmic oxide in the cornea , producing blindness.
◦ Inhibits hematoxylin and makes counterstaining difficult
◦ Extremely volatile
OSMIUM TETROXIDE (OSMIC ACID)

 Precautions
◦ Eyes and skin may be protected by using a fume
hood or wearing protective plastic masks or gloves.
◦ Should be kept in dark colored, chemically clean
bottle to prevent evaporation and reduction by
sunlight or organic matter.
◦ Should be placed in cool place or refrigerated to
prevent deterioration
◦ Addition of saturated aqueous mercuric chloride
solution will prevent its reduction with formation of
black deposits
OSMIUM TETROXIDE (OSMIC ACID)

◦ Black osmic oxide crystals may be dissolved in

cold water.
◦ To prevent contact with black precipitate formed
in the bottom of the jar, the tissues may be
wrapped in cotton gauze and suspended in the
fluid by means of a thread.
◦ Osmic acid-fixed tissues must be washed in
running water for at least 24 hour to prevent
formation of artefacts.
Flemming’s Solution

 Most common chrome-osmium acetic acid

fixative used, recommended for nuclear
preparation of such sections.
 Formula
◦ Aqueous chromic acid 1% ---------------------------15ml.
◦ Aqueous osmium tetroxide 2%------------------------4ml.
◦ Glacial acetic acid -----------------------------------1ml.
 Fixation Time ------24-48 hours
Flemming’s Solution

 Advantages
◦ Excellent fixative for nuclear structures
◦ Permanently fixes fat
◦ Relatively less amount of solution is required for
fixation (less than 10x the volume of the tissues
to be fixed)
Flemming’s Solution

 Disadvantages
◦ Poor penetrating agent
◦ Solution deteriorates rapidly and must be
prepared immediately before use
◦ Chromic-osmic aid combination depress the
staining power of hematoxylin
◦ Has tendency to form artifect pigments
◦ Very expensive
  
Flemming’s Solution without acetic acid

 Made up only of chromic and osmic 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.
 Fixation time - - 24-48hours
TRICHLOROACETIC ACID

 Sometimes incorporated into compound fixatives

 Advantages

◦ Precipitates proteins
◦ Its marked swelling effect on tissues serves to
counteract shrinkage produced by other fixatives.
◦ May be used as a weak decalcifying agent
◦ Softening effect on dense fibrous tissues facilitates
preparation of such sections.
 Disadvantages
◦ Poor penetrating agent
 
ACETONE

 Used at ice cold temperature ranging from

-5C to 4C
 Advantages

◦ Recommended for the study of water diffusible

enzymes especially phosphatases and lipases
◦ Used in fixing brain tissues for diagnosis of
rabies
◦ Used as a solvent for certain metallic salts to be
used in freeze substitution techniques for tissue
blocks
ACETONE

 Disadvantages
◦ Produce inevitable shrinkage and distortion
◦ Dissolves fat
◦ Preserves glycogen poorly.
◦ Evaporates rapidly.
  
HEAT FIXATION

 Procedure involves thermal coagulation of

tissue proteins for rapid diagnosis, usually
employed for frozen tissue sections and
preparation of bacteriologic smears.
 Advantages

◦ Fixation is better
◦ Preserve nuclear and cytoplasmic detail
◦ Suitable for frozen tissue preparation
HEAT FIXATION

 Disadvantages
◦ Produces considerable tissue shrinkage and
distortion
◦ Destroys RBC
◦ Dissolves starch and glycogen
SECONDARY FIXATION

 The process of placing an already fixed

tissue in a second fixative in order:
◦ To facilitate and improve the demonstration of
particular substances
◦ To make special staining techniques possible
(with secondary fixative acting as mordant)
◦ To ensure further and complete hardening and
preservation of tissues
SECONDARY FIXATION

 This may be done before dehydration and

on deparaffinized sections before staining,
usually with 10% formol saline as primary
fixative. The tissue may be placed in a
primary fixative for storage or may require
further fixation for special staining.
POST-CHROMATIZATION

 Form of secondary fixation whereby a

primarily fixed tissue is placed in aqueous
solution of 2.5 -3% potassium dichromate
for 24 hours to act as mordant for better
staining effects and to aid in cytologic
preservation of tissues.
 
WASHING OUT

 The process of removing excess fixative from the

tissue after fixation in order to improve staining and
remove artefacts from the tissues.
 Several solutions may be used:

◦ Tap water is used to remove:

 Excess chromates from tissues fixed in kelly’s, zenker’s
and flemming’s solutions
 Excess formalin
 Excess osmic acid
◦ 50-70% alcohol- used to wash out excess amount of picric
acid (Bouin’s solution)
◦ Alcoholic iodine – used to remove excessive mercuric
fixatives
Factors that affect fixation of tissues:
 Retarded by:
◦ Size and thickness of the tissue specimen-larger
tissues
◦ Presence of mucus – prevents complete
penetration of fixative
◦ Presence of fat
◦ Presence of blood
◦ Cold temperature – inactivate enzymes
Factors that affect fixation of tissues:
 Enhanced by:
◦ Size and thickness- smaller tissues
◦ Agitation – fixation is accelerated when automatic
or mechanical tissue processing is used
 Moderate heat (37-56C) accelerates fixation but
hastens autolytic changes and enzymes
destruction.
  
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Autopsy material should be fixed as soon after
death as possible to prevent decomposition and
autolysis due to deprived oxygen metabolism
 Surgical specimens should be fixed as soon as

possible after removal or refrigerated if fixation

is to be delayed, to prevent drying of surface
layers and ultimate tissue distortion.
 If placed in NSS during the operation, autolysis

may occur before fixation is carried on.

 All tissue specimens must be properly labeled

and identified
PRINCIPLES AND PRECAUTIONS IN HANDLING AND
FIXATION OF SPECIMENS IN GENERAL

 If tissues are refrigerated, slow freezing of unfixed

tissues near 0C must be avoided since this may
promote formation of ice crystal artefacts.
 Tissue slices should be taken at right angles to the
surface of the organ and should be sufficiently
deep to show the normal anatomic components.
 Tissues should not be more than 5mm thick except
in lung edema, with minimum squeezing and
handling.
 Thin sections allow complete penetration by fixative
in a short time.
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Purulent materials, exudates or exudates should be
marked and kept for possible cultures, smears and
other bacteriologic examination.
 The amount of fixative must be adequate (20times
the volume of specimen) except for osmium tetroxide
which is very expensive, requiring only 5-10 times
that of tissue volume for fixation.
 For prolonged fixation (museum preparation) the
volume of fixing fluid should not be less than 50-100
times that of the tissue.
 Contamination of fixed tissue with precipitates
should be avoided.
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 In most instances, fixed tissues must be washed
thoroughly to remove salts and or pigments before
staining.
 Low temperature retards fixation but prevents autolysis.
 The required period of fixation should not be exceeded

since this may cause excessive hardening or brittleness of

tissues.
 There must always be an adequate supply of fixatives at all

times.
 Drying should be avoided to prevent shrinkage and

distortion of tissue with loss of cellular detail. Small tissue

biopsies may be placed in a petri dish with moistened filter
paper to prevent drying.
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Hollow organs should be packed with cotton soaked
in fixative or completely opened before being
immersed in adequate fixing solution.
 Air filled lungs may float on fixative. To avoid this,
the organ may be covered with several layers of
gauze to maintain it under surface.
 Human brains may be suspended by a cord tied
under the circle of willis to prevent flattening, and
may undergo intravascular perfusion.
 Eyes must be dissected before they are fixed since
this may lead to immediate tissue collapse and
wrinkling to the escape of vitreous humor.
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Frozen sections may lead to formation of ice crystal
artifacts.
 To avoid rigor contraction and staining of artifacts when
fixing muscle, the fresh biopsy material may be stretch
for by sutures on each end, or laid flat in a moist filter
paper before suspending in fixative.
 Water should not be used for glycogen containing tissues
because glycogen is soluble in water.
 To assure rapid access of the fixative to all parts of the
tissue, the tissue may be minced, that is, small pieces of
specimen may be divided into small fragments and
transferred to the vial of a fixative by means of a
toothpick.
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Hard tissues may be washed out in running
water overnight and immersed in 4%
aqueous phenol solution for 1-3 days
(Lendrum’ method). This will soften the
tissue and allow easier sectioning without
producing any marked distortion of the cell
structures.
 Good cutting and staining of section is

greatly dependent upon proper fixation.

 
PRINCIPLES AND PRECAUTIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
 Proper tissue processing should start with proper
fixation and preservation since a badly preserved
tissue will later on yield a badly processed
specimen and may prove to be unsuitable for. The
choice of fixatives and mode of processing must
therefore vary depending upon the following
factors:
◦ Need for immediate examination
◦ Tissue structure or component to be studied
◦ Type of tissue to be processed
◦ Staining technique to be applied
◦ Type of section to be made, whether serial or individual.
 SOME OF THE DIFFICULTIES ENCOUNTERED
BECAUSE OF IMPROPER FIXATION ARE:
PROBLEM CAUSE

Failure to arrest early autolysis of cells Failure to fix immediately, insufficient

fixative
Removal of substances soluble in fixing Wrong choice of fixatives
agents
Presence of artifact pigments on tissue Incomplete washing of fixatives
sections
Tissues are soft and feather like in Incomplete fixation
consistency
Loss or inactivation of enzymes needed for Wrong choice of fixative
the study
Shrinkage and swelling of cells and tissue overfixation
structure
Tissue blocks are brittle and hard Prolonged fixation

An incompletely fixed tissue may lead to improper and incomplete clearing and
impregnation, and may later be a hindrance to normal sectioning and staining of
specimen.
FIXATION ARTEFACTS

 Formalin pigment is a well known artifact that

may be produced under acid conditions.
 The pigment may be eliminated or reduced by
fixation in phenol formalin.
 “Crush artifact” may be found in surgical
specimens particularly in liver biopsies,
associated with intense eosinophilic staining at
the center of the tissue. This may be due to
partial coagulation of partially fixed protein by
ethanol or by incomplete wax impregnation
during subsequent histological processing.
MICROWAVE TECHNIQUE

 Works as a physical agent similar in mechanism to

vacuum, oven (heat) and agitation to increase the
movement of molecules and accelerate fixation.
 Used to accelerate staining , decalcification,

immunohistochemistry, and electron microscopy.

 Interacts with dipolar molecules, causing oscillation at

a frequency of 2450 mHz

 Water molecules and polar side chains of proteins

increase their thermal energy, with subsequent

heating of the proteins.
 Allows light microscopic techniques used in routine

histopathology to be performed adequately.

MICROWAVE TECHNIQUE

 Advantages
◦ Chief advantage of microwave fixation is that the
tissue is heated right through the block in a very
short time, thereby potentially allowing the study
of cellular processes that proceed very rapidly
◦ Non-chemical technique that is useful in preserving
neurochemical substances in brain, such
acetylcholine.
◦ Used for rapid fixation of routine surgical
specimens.
◦ Significantly reduces the time taken for
immunohistochemistry and in-situ hybridization
MICROWAVE TECHNIQUE

 Disadvantages
◦ Microwaves generated by commercial ovens only
penetrate tissue to a thickness of 10-15mm.
◦ No significant cross-linking of protein
molecules, and subsequent chemical fixation may
be needed.
◦ Viable spores and other pathogens may remain in
tissues processes with alcohol-based fixatives or
microwaving alone.
  
MICROWAVE TECHNIQUE

 Fixation: procedure
◦ Fix tissue------------------------------4hours
◦ Soak blocks in water at room temp --------1 minute
◦ Place blocks in 100ml of formalin---------------
Place in microwave , 450watts,55C------1.5 to 4mins
◦ Remove blocks and slice tissue to 2mm thick
◦ Place directly in alcohol
 Dehydration can be achieved in one step
(instead of 6) in microwave, using ethyl or
methyl alcohol.
MICROWAVE TECHNIQUE

 Clearing agents – ethyl alcohol (dehydrant),

isopropanol (, best), histoclear,xylene,
chloroform, methyl alcohol
◦ Isopropanol absorbs microwave better than other
reagents , less toxic than xylene and can
effectively boil out at paraffin stage due to
boiling point.
◦ Cheaper than ethanol and help dehydrate further.
◦ It need only one change in paraffin bath, or 2
changes if dense tissue is being processed.
  
IMMUNOFLOURESCENCE AND IMMUNOPEROXIDASE
TECHNIQUES

 Immunoflourescence technique are

commonly used in pathology for the
demonstration of antibodies.
Enzyme histochemistry
◦ The general aim of fixation is to preserve the
maximum enzyme activity at its original
localization, while also preserving structural
integrity.
◦ The tissue for study may be fixed in 4 %
formaldehyde or formol saline overnight, or fresh
frozen cryostat sections may be fixed in acetone
or formaldehyde and washed in distilled water
prior to enzyme testing.
Electron microscopy

◦ The most useful fixative for EM are osmium

tetroxide, glutaraldehyde and paraformaldehyde,
with the whole procedure performed at 4C.
◦ For routine studies, glutaraldehyde or osmium
are adequate.
◦ For electron histochemistry and electron
immunocytochemistry karnovsky
paraformaldehyde-glutaraldehyde is useful.
  
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The end….