Fixation

FIXATION
 Why do tissues require fixation –
 To avoid decomposition due to deprivation of oxygen, accumulation of carbon
dioxide, metabolites
 To avoid putrefaction and action of various enzymes (autolysis)
 Putrefaction – tissues with high bacterial content (eg.
Gastrointestinal tract) undergoes rapid breakdown by the action
of organisms with the production of gas
 Autolysis – Tissue destruction caused by action of enzymes
released by lysosomes
 To maintain the tissue in a life like manner without change in their structure
 What is fixation
 Fixation is process in which cells or tissue are fixed in physical state and
partly in chemical state so that they will with stand subsequent treatment with
various reagents with a minimum loss, distortion or decomposition.
 What is the principle in the action of fixative
 Fixatives act by denaturing or precipitating proteins which then form a
meshwork due to cross linking of proteins. This meshwork tends to hold the
other cell constituents in vivo relation to each other and insoluble proteins
provide mechanical strength for subsequent procedures.
 What is an ideal fixative
 Ideal fixative should
 Penetrate tissue quickly
 Rapid in action
 Isotonic
 Cause minimum loss and minimum physical and chemical
alteration of the tissue and its components
 Cheap, stable and safe to use.
 What are the affects of fixatives on tissue
 Produces tissue hardening
 Some fixatives act as mordants for certain stains eg. Mercury fixatives
 Increases the optical differentiation of cells and tissue components
 Renders the cell insensitive to hypo and hypertonic solutions used subsequent
to fixation
 Microorganisms are killed (fixed) which will prevent putrefaction of tissues
and decreases the risk of infection to the person handling it
 There is no ideal fixative for all tissue but it differs from tissue to tissue.
 Amount of fluid to be used in fixing the specimen – 10 to 20 times volume of
specimen should be used except osmium tetroxide (used in electron microscopy)
 How do you preserve autopsy material
 Embalming the body
 Slices of organs not more than 5mm thick except lung where slices should be 1
to 2 cm in order to show edema

 Large organs if desired to preserve in toto should be preserved by vascular
perfusion with buffered neutral formalin or by slicing the organ at 1cm
intervals and immersing it in large volume of fixatives.
 How is the brain fixed
 Brain is best fixed by preautopsy embalming or after removal by injection via
the basilar artery followed by immersion for 1 to 3 weeks in large brain jar or
 pail of formalin. Distortion of the brain is prevented by suspending it in the
fluid by string or cotton thread passed under the arterial circle of willis.
 The brain may be sliced with a long bladed knife at 1 to 2cm intervals before
being placed in fixative
 Brain is placed in large plastic bags together with adequate fixative. The bags
are then sealed and placed in large tanks of water so that buoyancy will
prevent distortion
 How are the lungs fixed
 Lungs are fixed by intra tracheal or intra-arterial injection of buffered
formalin or formal saline with a container of formal saline being held 30 to 40
inches (75 to 100cms) above the canula
 Various methods of fixation
 Immersion/in vitro
 Perfusion/in vivo
 Vapour
 Spray/coating
 Freeze drying
 Microwave fixation
 Factors affecting fixation 
 Size and thickness of the piece of tissue
 Tissue covered by mucous or blood may have slow penetration of fixative
 Tissue containing fat is fixed slowly.
 PH-Satisfactory fixation occurs at PH 6 to 8
 Temperature – Usually at room temperature (for electron microscopy -0-4c)
 Osmality of fixative – Fixative should be isotonic
 Hypertonic solutions –Cell shrinkage
 Hypotonic solution – Swelling cells & poor fixation.
 Classification of fixatives into three main groups
 Group A –
 Microanatomical fixative
 Cytological fixative
 Histochemical fixative
 Group B-
 Coagulant fixatives
 Non coagulant fixatives
 Group C –
  Simple
 Aldehydes
 Oxidizing agents
 Protein denaturing agents
 Unknown mechanism
 Compound fixative
 Group A (Based on structure to be preserved)
 Micro anatomical fixative
 They preserve anatomy of tissues and various layers of tissues in
relation to each other
 Formalin based fixative
 Buffered Glutaraldehyde
 Zenker fluid, Zenkers formal
 Formal calcium
 Cytological fixatives-
  Used when intracellular structures and inclusions has to be
demonstrated .They are fixatives divided into
 Cytoplasmic fixatives –
 Do not contain glacial acetic acid.
 Reaction of hyaline occurs at pH 4.6 or higher
 Eg: Zenkers fluid
 Schaudinn fluid
 Regaud’s fluid
 Formal saline & Formal calcium
  Nuclear Fixatives –
 Usually contain glacial acetic acid because of the
affinity for it to nuclear chromatin
 .They usually have PH of 4.6 or less
 Eg: Carnoy’s fluid, Clarkee fluid,
Flemming’s fluid
 Zenkers, Formal saline and Formal calcium are good
cytoplasmic and microanatomical fixatives
 Histochemical fixatives:
 These fixatives aim at preserving the chemical constituents of
cells.
 Eg:   Formalin Saline, cold acetone, Absolute alcohol
 Group B:
 Coagulant fixatives: This fixative transforms the protein of the cytoplasm into
fine mesh which does not interfere with light microscopy but not used in
electron microscopy. They separate proteins from water as coagulum. 
 Ex. Mercuric chloride fixatives
 Non coagulant fixatives: They harden protein gels without separating the
water from the protein in the gel. These fixatives fix the cytoplasm without the
formation of the fine sponge like threads
 Ex: Formaldehyde
 Group C: (Based on the chemical nature)
 Simple fixatives –These fixatives are further classified as
  Aldehyde –
 Formaldehyde
 Glutaraldehyde
  Oxidizing agents –
 Osmium tetroxide
 Potassium dichromate
 Potassium permanganate
 Protein denaturing agents –
 Acetic acid
 Ethyl alcohol
 Methyl alcohol
 Unknown mechanism –
 Picric acid
 Mercuric acid
 Compound fixatives– These are the products of two or more simple fixatives
to obtain the combined affects
 Ex: – 10% Formal saline
 10% Buffered Formal
 Formal Calcium
 Zenker fluid
  Zenkers formal
 

 FORMALIN
 Commercial formaldehyde is saturated solution of formaldehyde (H.CHO) gas in water,
approximately 40% gas by weight.
 10% of formalin used for fixation is prepared by adding 10ml of formalin to 90ml of saline.
 Turbidity in the formalin is due to formation of paraformaldehyde which is formed due to
polymerization of formaldehyde. This may be removed by filtration. Usually commercial
formaldehydes contain 11% to 16% methanol which inhibits the formation of
paraformaldehyde.
 Formic acid formed in Formaldehyde reduces the quality of routine staining, particularly
nuclear, leaches out hemosiderin and promotes the formation of formalin pigment.This can
be prevented by using formal saline to which handful of calcium carbonate, then shaken
well and stored in jar containing a layer of marble chips.
  Principle of formalin fixation
 Formalin acts by polymerizing action, i.e, the formation of complexes by
development of links (methylene bridges) between protein molecules.
 Minimum time required for fixation
 8 hours at room temperature. 4 hours may be sufficient with agitation. Time
can be reduced to 25 to 40% by increasing the temperature to 450C.
 Average thickness of tissue for adequate fixation – 4mm
 Advantages of formalin fixation:
 Cheap & easy to prepare
 Allows the uses of various staining techniques
 Frozen sections can be prepared
 Fat stains can be used as formalin fixed tissues
 Does not cause excessive hardening of tissues
 Natural tissue colour can be restored without difficulty
 It is the best fixative for the nervous system.
 Disadvantages of formalin fixation:
 Can lead to dermatitis of the hands & irritation to the nostrils due to fumes
(can be prevented by proper ventilation).
 If excess of blood is present in tissues then formalin leads to formation of dark
brown artifact pigment granules which are doubly retractile.
 Methanol which is added to formalin to prevent the formation of para
formaldehyde causes denaturation of proteins& makes it unsuitable for
electron microscopy.
 Formalin pigment & its removal
  Formalin pigment is brown granular material formed by the action of
formalin in excess of blood. It is removed by
 Picric acid – Place the sections in the saturated alcoholic solution
of picric acid for 20 min to 2 hrs & then wash under tap water
for 10 to 15 min.
 Kardasewitsch’s method – After washing with water place the
sections in the following mixture for 5 min to 3 hrs then wash
with water
 7% ethylalcohol – 100ml
 20% Ammonia  –  10-20 ml

 Lillies method– After washing with water place the sections in
the following mixture for 1 to 5 mns then wash with water.
  Acetone (50ml)
 3 vol Hydrogen peroxide (50ml)
 28 % of ammonia water (1m)
 Different formalin based fixatives:
 10% formalin
 10% formal saline –
 Water -900ml
 Sodium chloride -8.5gm
 Formalin -100ml
 10% of buffered neutral formalin –
 Water -900ml
 NaH2Po4 (anhydrous) -3.5gm
 Na2HPo4 (anhydrous)-6.5gm
 Formalin -100ml
 Hydrated salts –
 NaH2Po4.H2O-4.02g
 Na2HPo4 .12H2O-16.37g/l
 Formal calcium (calcium acetate formalin) –
 Distilled water -90ml
 Calcium acetate monohydrate -2gm
 Formalin -10ml
 Secondary fixation (Post mordanting)
 It is sequential application of two fixatives to improve the staining character
of tissue
 Blocks which are in formalin for 1 to 4 hrs are placed for 4 to 6hrs in Helly’s
fluid or 4 to 16 hrs in formal mercuric chloride
 Formal Mercuric chloride
 Mercuric chloride -30g
 Distilled water -900ml
 Formalin -100ml
 Fixatives for electron microscopy
 Tissue should be fixed at 4oc in the refrigerator
 Tissue fragments should be ½ to 1 by 1mm
 Glutaraldehyde
 Osmum tetroxide
 Acetaldehyde acrolien
GLUTARALDEHYDE
 Stable at 0 to 4oc and at PH 3.0 to 5.0
 To remove the impurities in Glutaraldehyde which are polymers of glutaraldehyde (eg
Acrolein, Ethanol, Glutaric acid etc) Charcoal is added.
 For fixation 2.5 % to 4% conc. is required.
 Advantages
 Formation of more cross linkages with better preservation of cellular & fluid
proteins
 Resists acid hydrolysis
 Causes less shrinkage than formalin
 More pleasant & less irritant
 Does not corrode metal
 Does not cause dermatitis
 Disadvantages:
 Expensive
 Less stable
  Penetrates tissue more slowly from formalin
 Inferior formalin for PAS satin.
 

OSMIUM TETROXIDE (OsO4)

METALLIC FIXATIVES
 Mercuric fixatives
 Chromate fixatives
 Lead fixatives
Mercuric fixatives
 Principle– Mercuric ions act by combining with acidic (carboxyl –COOH) groups of
proteins & form especially strong combination with the sulfer (thiol) radicals
 Zenkers
 Helly’s Formal .Zenkers (Best for fixation of blood forming or blood
containing tissues i.e; spleen or bone marrow
 Heidenhain’s “Susa “fixative
 Schaudinn’s sublimated alcohol
 B5 fixative 
 Zenkers fixative
 HgCl2 – 50gm
 Potassium dichromate – 25gm
 Sodium sulphate – 10gm
 Distilled water – 1000ml
 Add 50ml glacial acetic acid before use (5 ml/dl of stock)
 Helly’s formal Zenker composition:
 Zenkers fixative + formalin 5-10ml to each 100ml of stock
 Heidenhain “Susa”composition:
 HgCl2 -45gm
 Nacl – 5gm
 Formalin (40% formaldehyde solution )- 200 ml
 Glacial acetic acid – 40 ml
 Trichloroacetic acid – 20 gm
 Distilled water – 800 ml
 Schaudinn’s sub limited alcohol composition:
 Hgcl2 -3 gm
 Distilled water – 50 ml
 B5 fixative
 Mercuric chloride – 12gm
 Sodium acetate – 2.5 gm
 Distilled water – 200 ml
 Formalin just before use – 2ml to the 20ml of above solution  
  Advantages of mercury fixatives:
 Better staining of nuclei and connective tissue
 Cytoplasmic staining –enhanced with acidic dyes.
 Nuclear chromatin shown in detail
 Preservation of details for photography.
 Best results with metachromatic stain
 B5 fixative is frequently used for bone marrow,spleen, lymph nodes and other
hematopoetic tissue
 Disadvantages of Hg fixatives:
 Corrodes the metals
 Lysis of RBC & removes much iron from hemosiderin
 Deteriorates rapidly
 Causes Marked  shrinkage
 Reduces the amount of demonstrable glycogen
 Slow penetration
 Tissues become hard & brittle
 Formation of Diffuse black granules in tissues
 Radiopaque: preclude use of x-rays to determine and point of calcification
 Precautions-Removal of black mercury granules
 Place sections in 70% alcohol to which scherald solution of iodine is added
(0.5ml +100ml alcohol iodine),less for 1 to 2 min to remove merurounchloride
deposit
 2Hgcl+I2 =Hgcl2+HgI2
 Rinse in water
 Place in 5% sodium thiosulphate for 1 to 2 min to remove Iodine
 2 Na2S2O3+I2 = 2 Na I+Na2S4O6
 Wash in running tap water to remove sodium thiosulphate crystals
 

Chromate fixatives
 Principle – Chromium salts in H2O form Cr-O- Cr complexes which have an affinity for
the COOH & -OH groups of proteins so that complexes between adjacent protein
molecular are formed.
 This leads to disruption of the internal salt linkages of the protein increasing the reactive
basic groups & thereby enhancing acidophilic in staining
 Eg  –Orth’s fluid
  Regauds fluid
 Orth’s fluid

 5% Potassium dichromate -100 ml
 Sodium sulphate – 1gm
 Add formalin before using – 10 ml

 Regaud’s (moller’s fluid)
 Potassium dichromate – 80ml
 Add formalin before using – 20ml
 Advantages:
 For demonstration of chromaffin tissues (eg: Adrenal medulla, mitochondria,
Golgi apparatus, mitotic figures & RBC’s)
 Best for preserving phospholipids
 Disadvantage of chromate fixative
 Prolonged fixation in chromate – Bleach all tissues pigments (melanin)
 
 Glycogen preservation is poor
 

Lead fixative
 Lead like other metal fixatives, precipitate proteins. They are used mainly for
mucopolysacharides
 Eg Lillies Alcoholic lead nitrate formalin
 lead subacetate
 Lillies alcoholic lead nitrate formalin
 Lead nitrate – 8gm
 Formalin – 10 ml
 Distilled water – 10 ml
 Absolute ethanol – 80 ml
 Lead sub acetate
 Lead sub acetate -4gm
 Co2 free distilled H2O-100 ml
 Glacial acetic acid to make clear solution – up to 2.0 ml
 Formalin optional – 10 ml
Other fixatives
 Picric acid fixatives
 It gives better preservation of alcohol
 Picric acid forms protein picrates, some of which are water soluble until
treated with alcohol
 Eg: Bouins fluid
 Gendres fixative
 Brasil’s alcoholic – picro formol fixative
 Bouin’s fluid–
 1.2% aqueous picric acid -75ml
 Formalin – 25ml
 Glacial acetic acid – 5ml
 Advantages –
 Good for demonstration of glycogen
 Best for testicular biopsies
 Penetrates rapidly
 Shrinkage is less
 Suitable for mallory’s, heidenhain’s & masson’s aniline stain.
 Due to picking up of yellow colour small tissues can be easily
identified.
 Disadvantages –
 Lyses RBCs & reduces the amount of demonstrable ferric iron
 If tissue left for longer than 12 to 24 hrs try become hard &
brittle
 Lipids are both altered & decreased
 The yellow colour of the sections of the slide is removed by 
 Place the sections in a saturated solution of lithium
carbonate in 70% ethylalcohol for few minutes.
 Treat the sections with ethylalcohol followed by 5%
sodium thiosulphate
 Wash in running tap water
 
 Alcoholic fixatives
  Alcohols denaturation and precipitates protein
 Methylalcohol
 Ethylalcohol
 Carnoy’s alcohol
 Clarke’s fluid
 Methylalcohol –
 80 to 100% used for smears either wet or dry
 below 80% -cell lysis occur
 Ethylalcohol –Fixative for enzymes
 Carnoy’s fluid
 Absolute ethylalcohol – 60 ml
 Choloform – 30ml
 Glacial acetic acid -10ml
 Clarkes’s fluid
 3 vol methanol plus 1 vol glacial acetic acid)
 Advantages:
 Carnoy’s fluid is best for small tissue fragments like curetting
 It is a good fixative for glycogen
 Nuclear staining & carbohydrate preservation are good.
 Clarke’s fluid is used as fixative for cell cultures in chromosome studies
 Disadvantages of alcoholic fixative
 Causes severe shrinkage (unless used at colder temperature)
 Hardens tissue excursively
 Lipids any myelin are dissolved
 At 5 to -20oc it preserves some enzymes like alkaline phosphatase
 Although good fixative for glycogen it leads to “polarization” because of the
streaming of the glycogen granules to one pole of the cells.
 Acetone –Acts parallel to alcohol & used in enzyme studies
Flemming fixative 
 1% aqueous chromic acid -15ml
 2% aqueous osmium tetroxide -4ml
 Acetic acid -1ml
 Suitable for electron microscopy & for myelin in peripheral nerves
Heat fixation
 Ether saline (0.85%) or 10% formal saline is used.
 20 to 40 ml is heated below the boiling point then the tissue slice (3 to 5mm thick) is placed
in hot fluid & heating is continued for 1 min until tissue floats to the surface.
 After this it is cooled quickly in water & mounted on microtome.
 Fixation of needle biopsies
 Small needle biopsies from organs such as kidney, liver & brain can be fixed
in
 Zenkers or Helly’s fluid –
 30 to 60 min fixation is sufficient
 It left for longer time hardens tissue & mass it over
oxidised destroying PAS reactivity.
 Buffered neutral formalin
 
 Muscle biopsy-
 To prevent the contractions & staining artifacts, fresh biopsy should be
stretched for 30min by means of sutures tied at each end (or) alternatively the
fresh biopsy to adhere to the piece of filling cord for 15 to 30 min
  Drying should be avoided hence the material should be placed in petridish
with moist filter paper as its floor
 After 15 to 30 mins the biopsy is placed in fixative
 One fixed two blocks are selected one with longitudinal section s one with
transverse section
 Softening of hard tissue (Lendrum method)
 Essential in case of cervix, fibroids, hyperlceratotic skin lesions & finger nails
 Procedure
 Washing the tissue in running tap water over night to remove the
fixative
 Tissue is then placed for 1 to 3 days in 4% aqueous phenol, after
which it is processes normally.
 Use of Benzene or choloroform instead of xylene will reduce
hardness
 Freeze drying:
 Use in histochemical studies
 Small pieces of tissue (1mm thick) are instantly frozen by immersion in
isopentane or propane –isopentane mixture cooled to -150oc with liquid
nitrogen. This prevents formation of ice crystals which can damage the tissue
 The tissue is rapidly transferred to a high vacuum drying apparatus kept at
about -40oc to -70oc
 When drying is complete (1 to 3 days) the tissue in embedded in paraplast.
 Then the specimen is quickly transferred to molten way (MP 56oc) in a
vacuum embedding oven and embedding is completed in less than 10 min
 Sections from freeze dried blocks must be affixed dry to albuminised slides or
floated out on warm mercury it they have to be processed unfixed or floated
out on 10 % formalin or formal calcium if fixation is required
 They should not be floated on water as it causes disintegration
 Permanganate fixation
 Suitable for preserving lipoprotein complexes Eg: cell membrane & myelin
SUMMARY
CLASSIFICATION OF FIXATIVES 
 

METALLIC FIXATIVES
 OTHER FIXATIVES

Reference 
1. Christopher Layton, John D. Bancrofti, S Kim Suvarna. Fixation of Tissues. In: Theory
and practice of histological techniques by John D. Bancrofti . 8th edition.
2. Cullings. Histotecniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch,
Stanely S. Raphael. Saunders publication 1983
3. Dr. Ganga S. Pilli. Practical Pathology.2007  
 4. Sabitri Sanyal, Aparna Bhattacharya.Clinical Pathology A practical Manual. 3rd edition.
2017. 
 

By –
 Dr. B. Syam Sundara Rao (Professor of Pathology, Narayana Medical College, Nellore)
 Dr. N.Mohan Rao (Professor of Pathology, Narayana Medical College, Nellore)
 Dr. V. Shanthi (Professor of Pathology, Narayana Medical College, Nellore)