Fixatives

By – Dr. Snigdha Das
Moderator–Dr.Veena Vidya Shankar

1. Introduction
2. Aim
3. Purpose
4. Effects of fixation
5. Reaction
6. Factors involved in fixation & methods
7. Classification of fixatives
8. Agents used in fixation
9. Secondary fixation
10. Post chromatization
11. Summary & References

Preparation of tissues for microscopic examination,
through a series of processes namely fixation,
dehydration, clearing, embedding, cutting and
staining.
Tissues decompose when removed from the
body or it is cut off from the blood supply –
Autolysis and Putrefaction

 Process by which the constituents of the cells
and therefore of the tissues are fixed in a
physical, partly also in a chemical state so that
they will withstand the subsequent treatment with
various reagents with minimum of loss ,
distortion or decomposition.
Fixation is the foundation for the subsequent
stages in the preparation of the sections

 Complex series of chemical events which differs
for different groups of substances found in
tissues.

 Dissecting table made up of hard wood with a working
surface area of 45 35 cm
 Wood- actual cutting surface
 Rubber pads of various dimensions- suitable
 Instrument- thin bladed knives 20-30 cm in length,
scissors, probes, scalpels, stainless-steel rule,
forceps, sponges
 First-aid box & eye-wash bottle
 Cutting board & instruments should be cleaned before
procedures
 Powdering of the gloves & washing of the gloved
hands before dissection

 Fixation of tissue- as soon as possible after death or
removal from the body
 Screw capped specimen jars containing appropriate
fixatives – in OT, post-mortem room, animal house
 Amount of fluid in the jars- 15-20 times
 Early dispatch of the specimens to histology laboratory
 Tissues should be washed in physiological saline
 Excessive blood & mucous should not be there
 Tissues selected for sectioning should be sufficiently
thin to be adequately fixed throughout a reasonable
time
 Best thickness- 3-5mm

 Preservation of cells &
tissue constituents in a
condition identical to that
existing during life.
 To do this in a manner that
will allow preparation of
thin, stained sections.

1. To prevent autolysis,
bacterial decomposition &
putrefaction.
2. To coagulate the tissue as
to prevent loss of easily
diffusible substances.
3. To fortify the tissue against
deleterious effects of further
stages.
4. To facilitate differential
staining with dyes & other
reagents.

1. Inhibition of autolysis &
putrefaction
2. Preservation
3. Hardening
4. Solidification of colloid
material
5. Optical differentiation
6. Effect on staining

 Produces immediate death of cells (life like
appearances)
 Prevents autolysis & putrefaction
 Reacts rapidly & completely with the tissue
 Fixes all the constituents of the tissue
 Neither shrinks nor swells
 Makes the specimen hard enough
 Raises refractive indices
 Provides full range of staining methods with great
selectivity

 No rigid upper limit of fixing time
 No tendency to deteriorate
 Easily prepared
 Cheap, safe to handle
 Non-toxic, stable
 Non-inflammable & non-irritant
 Should penetrate a tissue quickly
 Cause minimum physical and chemical
alteration of the tissue and its components.

 As such no ideal fixative.
 Choice depends on the cell or tissue
constituent to be demonstrated

1. Proteins:
 Cross links are formed
between proteins.
 Soluble proteins are fixed to
structural proteins- insoluble-
mechanical strength-allowing
subsequent maneuvers.
 Formaldehyde -reversible.
 Gluteraldehyde -rapid &
irreversible.
 React with basic amino-acid
residues

2. Nucleic acid:
 Fixation brings a change in the physical &
chemical state of RNA & DNA.
 Uncoiling of DNA & RNA occurs with formalin
when heated to 45˚C & 65˚C respectively.

3. Lipids:
 Phospholipids are fixed by aldehydes.
 Formaldehyde reacts with unsaturated fatty acids
hence less lipid can be demonstrated in tissue stored in
it for a long time.
 Mercuric chloride reacts with lipids to form complexes.
 Ultrastructural demonstration of lipids – post fixing in
imidazole-osmium tetroxide.

 Carbohydrates are more water soluble- difficulty
in total preservation
 They bind with fixed protein
 So the fixatives which are used for proteins, can
be used for carbohydrate preservation.
 Fixed protein traps carbohydrates.
 Glycogen not bound to protein- fixed protein
form lattice around glycogen to preserve it
 Glycogen are more demonstrable in liver cells

1. Buffers & hydrogen ion
concentration:
 Best fixation occurs between pH
6-8
 Buffers used – phosphate, s-
collidine, bicarbonate,
2. Temperature:
 Most tissues fixed– room temp
 Electron microscopy &
histochemistry – 0-4˚C
3. Penetration of tissues:
 Blocks should be small or thin to
ensure adequate penetration.

4. Volume changes:
 Due to inhibition of respiration, membrane
permeability changes, changes in ion transport
through membrane.
 Tissues fixed in formaldehyde & embedded in
paraffin shrink by 33%
5. Osmolality of fixative:
 Hypertonic solutions – cell shrinkage
 hypotonic solutions – swelling of cells & poor
fixation.
 Best – slightly hypertonic solutions.

6. Substances to vehicle:
 Adding substances to fulfill certain functions.
 Denaturing effects, some stabilize proteins.
 Eg. Sodium chloride & sodium sulphate
used with mercuric chloride.
 Tannic acid enhances fixation of lipids &
proteins in EM
7. Concentration of fixatives:
 Different concentrations have different
effects on morphology.
 Effects subsequent staining

8. Duration of fixation:
 Formalin – 2-6hours
 Electron microscopy – 3 hours
 Formaldehyde – prolonged fixation – shrinkage &
hardening of tissue.
 Gluteraldehyde – prolonged fixation – advantageous.
 Long fixation in aldehydes - inhibits enzyme activity.
 Long fixation in oxidizing fixatives – degrade the
tissue.

 Immersion/ in vitro fixation
 Perfusion/ in vivo fixation
 Perfusion-immersion
 Heat fixation

1. Aldehydes: formaldehyde, gluteraldehyde, acrolein
2. Oxidizing agents: osmium tetroxide, potassium
permanganate, potassium dichromate
3. Protein denaturing agents: acetic acid, methyl
alcohol, ethyl alcohol
4. Other cross linking agents: carbodiimides
5. Physical: heat, microwave
6. Unknown mechanism: mercuric chloride, picric
acid
7. HOPE fixatives: formalin like morphology, good
protein antigenicity for enzyme histochemistry, good
for RNA & DNA yeilds
General classification

 Aldehydes (cross linking agents)- act by creating
covalent chemical bonds between proteins of tissues-
anchor the insoluble compound to cytoskeleton-protect
secondary as well as tertiary structure of protein-
provide mechanical strength/ additional rigidity to
tissue structure.
 Oxidizing agents- joins with various side chains of
protein molecules & other biomolecules- allow
formation of cross link- stabilizes tissue structure

 Protein denaturing agents: reduce the solubility of
protein without combining with it & disrupts the
hydrophobic bonds which is needed for its tertiary
structure to form.
 Mercurials (B5 fixatives): it increases the staining
brightness & give good nuclear detail. Good for
reticulo-endothelial tissue & haemo-poetic tissue.
 Picrates:binds with histone & basic proteins to form
crystalline picrates with amino acid & precipitates
protein.

 Gas soluble in water up to
40% by wt.
 Available as 40%
formaldehyde or formalin.
 Stabilizer – 10-14%
methanol
 10% formalin
 Acidic solution.
 On storage becomes
acidic by formation of
formic acid.
 Colourless.
 Turbid on keeping -
paraformaldehyde.

 Yellow – contaminated
with ferric iron from metal
containers. Positive
prussian blue reaction.
 fixes protein, lipids well
preserved.
 Favors staining of acidic
structures like nuclei with
basic dyes
 Diminishes effect of acid
dyes on basic structures
like cytoplasm.

 Formalin is cheap
 Easy to prepare
 Relatively stable
 Frozen sections can be prepared with ease.
 Staining of fat and tissue enzymes.
 Penetrates tissues well.
 Beneficial hardening with little shrinkage

 Natural tissue colors are retained.
 Does not require washing before processing.
 Best fixative for nervous system

Disadvantage:
unpleasant vapour
irritation to eyes &
respiratory epithelium
Formalin dermatitis

 Brown, granular material,
extracellular, birefringent
 Progressive in deposition
 Often seen after several
days
 Action of acid formalin on
blood
 Avoided by using
buffered formalin.
 Removed – treatment
with saturated alcoholic
solution of picric acid for
20mins

 Peculiar artifact seen in sections fixed in formal saline &
stained with H&E.
 Complete or partial failure of nuclei to stain with
haematoxylin – take up eosin – loss of nuclear margin
distinction.
 Lymphoid & epithelial tissue – most distinct
 Patchy distribution
 Avoided by using - 2% acetic acid in 10% formalin
 When present – treat with 1% hydrochloric acid in
absolute alcohol for 1 hour before staining with H&E.

 Used for Electron
Microscopy with osmium
tetroxide
 Advantage:
 Most efficient cross linking
agent for collagen
 More rapid fixation than
formalin.
 Disadvantage:
 Poorer penetration
 False positivity with PAS
 More costly

 White crystalline substance.
 Powerful protein precipitant,
fixes both nucleus &
cytoplasm well favoring its
staining.
 Conjunction with other
fixatives.
 Adv: rapidly penetrates &
hardens tissue, radio-opaque
 Disadv: Extremely
poisonous & corrosive to
metals.
 Intolerant fixative.
 Pollution to environment.

 Mercury pigment –
brown to black granular
deposit.
 Treatment-
1. Place section in 0.5%
iodine in 80% alcohol for
3mins
2. Rinse in water
3. Place in 3% aqueous
sodium thiosulphate for
3mins
4. Wash in running water
for 1-2mins

 Bright yellow
crystalline
substance.
 Damped with water
because of
explosive
properties.
 Enhances
cytoplasmic
staining.
 Acts as mordant
 Much shrinkage but
little hardening.

 Orange crystalline
substance
 Less acidic pH – fixes
cytoplasm &
mitochondria
 More acidic pH – fixes
nucleus & cytoplasm
 Mordant
 Wash in running water
after to prevent formation
of insoluble precipitate.
 Prolonged exposure
causes tissue to become
brittle.

 Dark red crystals of
anhydride.
 Powerful oxidizing agent,
 Requires washing with
running water.

 Pale yellow.
 Demonstrates lipid like
myelin.
 Excellent preservation of
detail of single cells hence
used for EM.
 Uneven penetration for
pieces more than 2-3mm
 Storage in dark, cool place
 Vapour is irritating, causes
conjunctivitis
 Uneven fixation

 Colour-less liquid with
pungent odour.
 Used in compound fixatives
 Swells collagen fibres
 Precipitates nucleoproteins
 Solvent action on
cytoplasmic granules.

 Colour-less.
 Powerful dehydrating
agent.
 Causes shrinkage &
hardening
 Coagulates protein but not
nucleoprotein.
 Precipitates glycogen.
 Used in histochemical
method for enzymes.

Baker classification
Coagulant fixatives includes:
1.Formaldehyde
2.Gluteraldehyde
3.Osmium Tetroxide
4.Potassium Dichromate
5.Acetic Acid
Non-Coagulant fixatives includes:
1.Alcohol
2.Zinc salts
3.Mercuric chloride
4.Chromium trioxide
5.Picric Acid

1. Micro-anatomical fixatives:
When anatomy of tissues with correct relationship of
tissue layers & large aggregate of cells is to be
preserved.
2. Cytological fixatives:
To preserve constituent elements of cells.
Elements being preserved at the expense of
penetration, ease of cutting & loss of other cell
structures.
3. Histochemical fixatives:
When histochemical tests are to be applied.

I. Routine formalin fixatives:
1. 10% Formol-Saline:
 10% formalin in 0.9% sodium chloride.
 Layer of marble chips/calcium carbonate added to
neutralize formic acid production.
2. Buffered Formalin:
 Formalin – 10ml
 Acid sodium phosphate monohydrate 0.4g
 Anhydrous disodium phosphate 0.65g
 Water to 100ml

3. Formol Calcium(Baker)
 Calcium chloride – 2g
 Water – 100ml
 Chloride preserves
phospholipids
4. Formol Calcium(Lillie)
 Acetate instead of chloride
 Easily prepared
 Widely used for routine
fixation.

II. FORMALIN FIXATIVES FOR CARBOHYDRATES:
1. Buffered Gluteraldehyde
2. Heidenhain’s Susa:
 Mercuric chloride – 4.5g
 Sodium chloride – 0.5g
 Trichloroacetic acid – 2g
 Acetic acid – 4ml
 Distilled water to 100ml
 Adv – Excellent fixative for routine biopsy work.
 Brilliant staining with good cytological detail.
 Rapid penetration, minimum shrinkage
 Disadv – intolerant fixative
 Transfer to absolute alcohol is required

3. Zenker’s fluid:
 Mercuric chloride – 5g
 Potassium dichromate – 2.5g
 Sodium sulphate – 1g
 Distilled water to 100ml
 Glacial acetic acid immediately before use – 5ml
 Adv – rapid & even penetration
beneficial effect on cytologic, nuclear
chromatin & fibre stain
 Wash in running water.
 Fixation complete in 12hours

 Distilled water 100 ml
 Potassium dichromate 2.5g
 Sodium sulphate 1g
 Mercuric chloride 5g
 5ml of 40% formaldehyde before use
 Formalin instead of acetic acid.
 Excellent fixative for bone marrow, spleen
 Aka Spuler’s or Maximow’s fluid

7. Bouin’s fluid:
 Picric acid – 75ml
 Glacial acetic acid – 5ml
 Rapid & even peneration
 Fixed tissue gives brilliant
staining with trichome
methods
 Used to demonstrate
glycogen.
 Good for GIT biopsies

5. Gendre’s Fluid – good glycogen fixation
6. Rossman’s Fluid – carbohydrate fixation

I. NUCLEAR FIXATIVES:
1. Carnoy’s Fluid:
 Absolute alcohol – 60ml
 Chloroform – 30ml
 Glacial acetic acid – 10ml
 Penetrates very rapidly
 Excellent nuclear fixation
 Preserves Nissl substance&
glycogen
 chromosomes
 Destroys cytoplasmic elements.
 Rapid fixative – urgent
diagnosis.

2. Flemming fluid:
 Only 5-10 times of tissue bulk is required.
 Used as secondary fixative for myelin following
primary formalin fixation.
3. Newcomer’s fluid:
 Fixation of chromosomes
 Preserves chromatin better than Carnoy’s.

II. CYTOPLASMIC FIXATIVES:
1. Champy’s fluid:
 Should be freshly prepared.
 Poor & uneven penetration
 Preserves mitochondria, fat, yolk, lipids
 Preferred for mitochondria
2. Regaud’s fluid – mitochondria & chromaffin
tissue
3. Muller’s fluid – bone specimens
4. Zenker Formol
5. Schaudinn’s fluid – For wet smears

 Preserve the constituent to be demonstrated & its
morphological relationships.
 Without affecting the reactive groups & reagent to be
used in its visualization.
 Best – cryostat cut sections of rapidly frozen tissue.
 Formol saline
 Cold acetone – where enzymes are to be studied
especially phosphatases
 Absolute alcohol
 Vapour fixatives like formaldehyde, acetaldehyde,
gluteraldehyde, acrolein to fix cryostat cut sections of
fresh tissue & blocks of frozen dried tissue

 Urgent paraffin section
Thin slices are fixed in alcohol containing
fixatives Carnoy’s , formol alcohol
 Enzyme Histochemistry-
Fix in cold formol calcium
 Transmission Electron Microscopy
Osmium tetroxide, glutaraldehyde.
 Electron microscopy - 2% glutaraldehyde
 Immunoflourescence – unfixed cryostat

tissue Time interval fixative
Adrenal gland 1 or 2 hr Formaldehyde
eye Immediately after
death or within 2
hrs
Formol saline
Alimentary tract Immediately after
death
Susa fixative
Blood forming organ -do- Zenker’s fluid
Testis/ovary -do- Susa fixative
Lung/kidney/bone Fairly resistant to
postmortem
changes
Susa fixative
Renal biopsy Neutral buffer
formalin

Target Fixative of Choice
Proteins Neutral Buffered Formalin
Enzymes Frozen Sections
Lipids
Frozen Sections*,
Glutaraldehyde/Osmium
Tetroxide
Nucleic Acids Alcoholic fixatives, HOPE
Mucopolysaccharides Frozen Sections
Biogenic Amines
Bouin Solution, Neutral
Buffered Formalin
Glycogen Alcoholic based fixatives

 Tissues fixed with 2 fixatives in succession.
 Improved preservation & staining.
 Tissues fixed in buffered formalin – fixed with mercuric
chloride.
 Tissues fixed with gluteraldehyde is post fixed with
osmium tetroxide for electron microscopy.

 Treatment of tissues with 3% potassium dichromate
following normal fixation.
 Before processing – tissue in dichromate solution for 6-
8days
 After processing – for 12- 24 hrs
 Both followed by washing in running water
 Employed to mordant tissues.
 Mitochondria & myelin demonstrated.
 Improved preservation and staining of elements.

Is fixation always necessary????

 Formalin is used for all routine surgical
pathology and autopsy tissues when an
H & E slide is to be produced.
 Formalin is the most forgiving of all
fixatives when conditions are not ideal.
 B5 fixatives are recommended for reticulo-
endothelial tissues including lymph
nodes, spleen, thymus, and bone marrow.

 Bouin's solution is recommended for fixation
of testis, general fixative for connective
tissue stains.
 Glutaraldehyde is recommended for fixation of
tissues for electron microscopy.
 Alcohols, specifically ethanol, are used primarily
for cytologic smears.

 Bouin’s fluid- embryo fixation
 Commercially 40% by volume
 37% by weight
 Dessication- to study inorganic components
 Post fixation- softening by 4% phenol for 3-4
days- for better cut sections of tumours
 Secondary fixation- to prevent hardening effect
 Phenoxitol- ideal fixative, but very costly

 Carleton’s histological technique
 Histology, Microscopy & Photomicrography by Dr
D.R. Singh
 Histopathological & histochemical techniques-
C.F.A. CULLING

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