FIXATION

&
FIXATIVES
DR. NAMRATA SENGUPTA

PG 1 (ORAL PATHOLOGY)
GLOSSARY OF TERMS
AUTOLYSIS: lysis or dissolving of cells by enzymatic action, probably as a result of rupture
of lysozymes. The group of enzymes – cathepsins

PUTREFACTION: breakdown of tissue by bacterial action, often with the formation of gas.

DENATURATION: a major change from the original native state without alteration of the
molecule’s primary structure.

COAGULANT: it will allow solutions to penetrate into the interior of the tissue very easily.

NON COAGULANT: they act by creating a gel like barrier that makes the solution more
difficult to penetrate into the interior of the tissue.

PRECIPITATION: a process in which a solid is separated from a suspension or solution.

INTRODUCTION
 Once the tissue is removed from the body it will go through a
process of self destruction(AUTOLYSIS).

 If the tissue is left without any preservation, bacterial attack

will occur(PUTREFACTION).

 For proper examination & study of the tissue this has to be

prevented.
DEFINITION:
FIXATION:-
“ A process by which the constituents of cells or tissues are fixed in a physical
& chemical state so that they will withstand subsequent treatments with
various reagents with a minimum loss, distortion or decomposition.”

FIXATIVE (DORLAND’S):-
“ A fluid, often a mixture of several reactive chemicals, into which histological
or cytological specimens are placed so that, by processes such as
denaturation & cross-linking of proteins, autolysis is prevented, the specimen
is hardened to withstand further processing & the specimen is preserved in a
close facsimile of the living state in regard to both cellular morphology & the
location of subcellular constituents.”
 PURPOSE OF FIXATION
Fixation preserves a sample of biological material(tissue or cells) as close to its
natural state as possible in the process of preparing tissue for examination.
FEATURES:
First, a fixative usually acts to disable intrinsic biomolecules- particularly
proteolytic enzymes- which would otherwise digest or damage the sample.

Second, a fixative will typically protect a sample from extrinsic damage.

Fixatives are toxic to most commom microorganisms(bacteria in particular) which
might exist in a tissue sample or which might otherwise colonise the fixed tissue.
Finally, fixatives often alter the cells or tissues on a molecular level to
increase their mechanical strength & stability.

This increased strength and rigidity can help preserve the morphology(shape &
structure) of the sample as it is processed for further analysis.
IDEAL FIXATIVE
1) Produce immediate cell death(life-like appearances) without
shrinkage or swelling or distortion.
2) Penetrate the tissue rapidly & evenly.
3) Prevent autolysis & putrefaction.
4) Harden the tissue & render it insensitive to subsequent
treatment as staining.
5) It should allow the tissue to store for long time.
6) Raise the refractive indices.
7) Permit the restoration of natural colour for photography.
8) Simple to prepare & economical in use.
TYPES OF FIXATION
1) PHYSICAL FIXATION
 Heat fixation – simplest form of fixation
 Microwave fixation – speeds fixation; reduces time of fixation from >
12hrs to < 20 mins
 Freeze drying

2) CHEMICAL FIXATION
 Utilizes organic or non organic solutions to maintain adequate
morphological preservation.
 Chemical fixatives: coagulant, cross-linking, compound
BAKER’S CLASSIFICATION
1) COAGULANT FIXATIVES
 Alcohol
 Acetone
 Picric acid
 Trichloro acetic acid

2) NON COAGULANT CROSS-LINKING FIXATIVES

 Formaldehyde
 Glutaraldehyde
SIMPLE FIXATIVES
FORMALDEHYDE
i. Most commonly used fixative
ii. Gas soluble in water upto 40% by weight
iii. 40% formaldehyde gas in 100w/v of distilled water, resultant
mixture is 100% formalin
iv. Routinely 10% formalin used( 10ml of 100% formalin with
90ml of distilled water)
v. Fixes proteins, lipids
vi. Favours staining of acidic structures like nuclei with basic
dye
 TIME REQUIRED FOR FIXATION

At room temperature – 12hours

For small biopsies – 4 to 6 hours
At 65 degrees fixation occurs in 2hours
 ADVANTAGES
 Cheap
 Easy to prepare
 Relatively stable
 Frozen sections can be prepared with ease
 Staining of fat & tissue enzymes
 Penetrates tissue well
 Beneficial hardening with little shrinkage
 Natural tissue colour retained
GLUTARALDEHYDE
i. Used for electron microscopy with osmium tetroxide

ADVANTAGES
a) Most efficient cross-linking agent for collagen
b) More rapid fixation than formalin
DISADVANTAGES
a) Poor penetration
b) More costly
MERCURIC CHLORIDE
i. White crystalline substance
ii. Powerful protein precipitant, fixes both cytoplasm &
nucleus well favouring its staining
iii. ADV: rapidly penetrates & hardens tissues
iv. DISADV: extremely poisonous, corrosive to metals
PICRIC ACID
i. Bright yellow crystalline substance
ii. Enhances cytoplasmic staining
iii. Much shrinkage but little hardening
POTASSIUM DICHROMATE
i. Orange crystalline substance
ii. Less acidic pH – fixes cytoplasm & mitochondria
iii. More acidic pH – fixes nucleus
iv. Wash in running water after fixation to prevent formation
of insoluble precipitate.
OSMIUM TETROXIDE
i. Pale yellow
ii. Excellent preservation of details of single cell
hence used for EM
iii. Uneven penetration for pieces more than 2-3mm
iv. Store in dark cool place
v. Vapour is irritating causes conjunctivitis
ACETIC ACID
i. Colourless liquid with pungent odour
ii. Swells collagen fibres
iii. Precipitates nucleoproteins
 COMPOUND
FIXATIVES
A) FORMAL SALINE
FORMALDEHYDE(40%) – 100ml
SODIUM CHLORIDE – 9gm
DISTILLED WATER – 900ml
It is cheap & penetrates rapidly
Does not overharden the tissue

B) NEUTRAL BUFFERED FORMALIN

FORMALIN – 100ml
ACID SODIUM PHOSPHATE MONOHYDRATE – 4gm
ANHYDROUS DISODIUM PHOSPHATE – 6.5gm
WATER – 1000ml
Prevent formation of formalin pigments
PURPOSE
 The purpose of this study was to show the effect of formalin fixation

on surgical margins.
 Causes shrinkage of surgical margins which can result in the

underestimation of tumour-free margins.

MATERIAL & METHOD
 Cross-sectional study

 Study sample consisted of OSCC specimens of the GBS after

composite resection( 15 patients – 7men 8 women)

 All patients underwent composite resection & reconstruction with a

pectoralis major myocutaneous(PMMC) flap

 The primary predictor variable was the length of the linear
margin at various locations( anterior, posterior, medial &
lateral).
 All measurements were performed immediately after resection

by a single observer, using a digital vernier caliper.

 The primary outcome variable was the percentage of change

in each respective margin after fixation in 10% neutral

buffered formalin for 24hrs.
 Measurements after fixation were decreased compared to with

those taken before fixation

 The pathologist measures the specimen only after it has been
fixed and reports it accordingly. This can lead to a misleading
diagnosis and status of the margins.
 LIMITATIONS: Study carried out in a low volume institution, so

the sample is small. Studies using larger samples are needed

to quantify these findinds.
 Surgical specimens are routinely fixed in 10% NBF.
 A study compared the histomorphological and molecular assessments of formalin & ethanol
based fixatives.
 Mouse tissues were fixed in one of the three different fixatives (70% Ethanol, BE70 or 10%
NBF).
 BE70: 70% ethanol + 1% glycerol + 0.5% glacial acetic acid.

 For varying fixation times ( 4hr, 12hr, 1day, 1week, 1month, 3months, 6months).

RESULTS: 1. The quality of histomorphology and IHC in BE70 was relatively time-independent,

whereas those in NBF rapidly decreased after 1 week of fixation.

2. The quality & quantity of RNA extracted from tissue in ethanol based fixative showed
minimal changes from 4hr to 6months, whereas NBF had a dramatic detrimental change in RNA
quality after 1week of fixation.

3. Ethanol based fixative offers a superior DNA template for PCR amplification-
based molecular assays than NBF.

This comparative study was done by Joon-Yong Chung et al. in 2017.

C) ZENKER’S FLUID
Mercuric chloride 5g
Potassium dichromate 2.5g
Sodium sulphate 1g
Distilled water to 100ml
Add immediately before use glacial acetic acid – 5ml
 Good routine fixative

 Gives rapid & even penetration

NOTE:- Washing of tissue in running water is done to remove

excess dichromate
D) BOUIN’S FLUID
Picric acid saturated aq solution 75ml
Formalin (40% formaldehyde) 25ml
Glacial acetic acid 5ml
 Gives good nuclear details
 Good fixative for glycogen
 Excess picric acid is removed by alcohol treatment
E) CARNOY’S FLUID
Absolute alcohol 60ml
Chloroform 30ml
Glacial acetic acid 10ml
 Excellent NUCLEAR fixation
 Good fixative for carbohydrate
 Nissel’s substance & glycogen are preserved
 It dissolves cytoplasmic elements
 Fixation complete in 1-2 hours
F) CHAMPY’S FLUID
3% potassium dichromate 7ml
1% chromic acid 7ml
2% osmium tetroxide 4ml
 Penetrates poorly & unevenly
 Preserves mitochondria, fat & lipids
G) COLD ACETONE
 Action almost identical to alcohol.
 Glycogen is not well preserved.

 More recently acetone has been employed as a fixative in acetone-

methylbenzoate-xylene technique

H) ABSOLUTE ALCOHOL
 Sections cut by freezing technique can be fixed by absolute
alcohol for 24hrs
 Expensive
 SPRAY FIXATIVES
 Alcohol based
 Available in aerosol spray cans
 Fix cell smears on slides
 Contain water soluble wax – barrier against contamination

VAPOUR FIXATIVES
 Fix cryostat cut sections
 Formaldehyde – heating paraformaldehyde 50degree-80degree
 Acetaldehyde – 80 degree celsius for 1-4 hrs
 Glutaraldehyde – 80 degree celsius for 2mins-4hrs
POST / SECONDARY FIXATION
A second fixative is used. e.g Mercuric chloride for
4hrs after the tissue is being fixed by buffered
formaldehyde.

 It provides firmer texture to tissues.

 It also accentuates the staining.

REACTION OF FIXATIVES
1) PROTEINS
 Most important reaction which stabilizes proteins is by forming cross-links
between soluble protein & structural protein.
 Provide mechanical strength.
ALDEHYDES:-
a. Cross-links are formed between protein molecules & aldehyde group of
fixatives.
b. Aldehydes react with the basic amino acid residues of proteins.
c. Process takes place in 2 steps:
1st step small polymers are formed
2nd step small polymers cross-link
 Slow reaction
 Reversible
 Not good morphological picture
 Less effective at cross-linking
 Rapid
 Irreversible
 Good morphological picture
 More effective at cross-linking

FORMALDEHYDE GLUTARALDEHYDE
 OXIDIZING AGENTS:-
a) React with protein, form cross-links
b) Osmium tetroxide is more reactive towards protein

MERCURIC CHLORIDE:-
c) Reacts with histidine residues in proteins

HEAT/MICROWAVE FIXATION
d) Reacts with polar side chains – increases their thermal energy –
cause denaturation of proteins
2) NUCLEIC ACID

 Fixation brings about change in physical or chemical state of DNA or

RNA at room temperature.
 Few fixatives react with nucleic acid chemically – mercury, chromium
salts
 Heating at 45 & 65 degrees with Aldehyde fixatives, there is
uncoiling of RNA & DNA respectively.
 DNA is largely collapsed in methanol & ethanol.
 3) LIPIDS
 Most lipids are labile. So lost during routine processing. To
demonstrate them frozen section or cryostat is used.
i. ALDEHYDE FIXATION:- Preservation of lipoproteins. e.g
phospholipids which contain amino group are fixed by aldehyde.
ii. MERCURIC CHLORIDE react with highly unsaturated compound to
form complex.
iii. Ultrastructural demonstration – post fixation with osmium tetroxide.
iv. Cholesterol may be fixed with Digitonin for Ultrastructural
demonstration.
4) CARBOHYDRATE

 Single fixative – not satisfactory

 Alcoholic or picric acid fixatives – preservation of glycogen
 Ultra structural studies – glutaraldehyde is satisfactory
FACTORS AFFECTING FIXATION
I. BUFFER & pH

 Fixation is best carried out close to neutral pH, in the range of 6-8.
 Stabilization of tertiary & quaternary structure of proteins.
 By addition of acids – pH decreases – destruction of proteins & cause
precipitation.
 Hence, fixatives must be neutralized by adding buffers.
 Commonly used buffer system – phosphate, bicarbonate,veronal
acetate.
 Commercial formalin is buffered with phosphate at a pH of 7.
II. TEMPERATURE
 Most tissue fixed at room temperature.
 Electron microscopy & histochemistry: 0-4 degree celsius required.
 Increasing the temperature will increase the speed of fixation.
 Low temperature – slows down autolysis – more accurate details.
III. PENETRATION
 Fixation depends on the diffusion of fixative into the tissue.
 Penetration of fixative is a slow process.
 Size of specimen is important to ensure complete penetration of
fixatives.
 Small or thin slices of blocks – satisfactory fixation
 Large blocks – slow fixation
 d = k√t
d= depth penetrated
k=coefficient of diffusibility
t=time
IV. OSMOLALITY
 Hypertonic solutions – cell shrinkage
 Isotonic & hypotonic solutions – cell swelling
 By varying osmolality, structure of membrane system within various
cells can be altered.
Thus, additives to fixatives can alter extracellular space in
tissues.
 Sucrose is commonly added to osmium tetroxide for ultra structural
studies.
 Fixative solutions must be preferably isotonic, thus cell swelling is
compensated by processing & wax impregnation.
V. CONCENTRATION
 Low concentration of fixative with neutral pH favors fixation.
 Glutaraldehyde solution is used as 3% solution but it is effective
even at concentration as low as 0.05% with correct pH of fixative.
 Presence of buffer causes polymerization of aldehyde with a
consequent decrease in effective concentration.
 Staining of tissue is altered with the concentration of fixative
employed.
VI. DURATION
 Formalin : 2-6 hrs
 Electron microscopy : 3hrs
 Formaldehyde – prolong fixation – shrinkage & hardening of tissue
 Glutaraldehyde – effective polymer formation, advantageous
 Long fixation in aldehyde – inhibit enzyme activity & immunological
reactions
 Long fixation in oxidizing fixatives – degrade the tissue by oxidative
cleavage of proteins & loss of peptides
VII. OTHER FACTORS

A) VOLUME CHANGES
 Tissues commonly change in volume during fixation.
 Intercellular substance like collagen swell when fixed.
 Nuclei in frozen section is usually bigger than those subjected
to conventional preparation.
 Prolonged fixation in formalin causes secondary shrinkage.

B) SUBSTANCES ADDED TO VEHICLE

 Fixative solution = fixative agent+ buffer+water
 Salts added have denaturing & stabilizing effect on proteins.
FIXATION ARTEFACTS
 Something observed in a scientific investigation or experiment
that is not naturally present but occurs as a result of the
preparative or investigative procedure
 Putting some tissue in a fixative
 In fixatives containing mercuric chloride, a crystalline or

amorphous greenish-brown artifact pigment of mercury is

randomly deposited in tissues
- FALSE LOCALIZATION: due to diffusion of unfixed material.
Eg: ‘streaming effect’ (glycogen)
- FORMALIN PIGMENT: Brown granular material, extracellular.
Action of acid formalin on blood. Avoided by using buffered
formalin. Removed – treatment with saturated alcoholic solution
of picric acid for 20mints.
Glycogen Streaming artefact in a Formalin fixed section of
Liver stained by the PAS method
A Formalin-fixed Paraffin
section of Kidney showing
typical depositon of acid
formaldehyde hematin-
Formalin pigments.
SUMMARY
1) Formalin is used for all routine surgical pathology & autopsy
tissues when an H&E slide is to be produced.
2) Formalin is the most forgiving of all fixatives when
conditions are not ideal.
3) B5 fixatives are recommended for reticuloendothelial tissues
including lymph nodes, spleen, thymus & bone marrow.
4) Glutaraldehyde is recommended for fixation of tissues for
Electron Microscopy.
5) Alcohols, specifically ethanol, are used primarily for cytologic
smears.
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