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
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)
Used in electron microscopy
Used in fixing material for ultrathin sections for electron microscopy
Disadvantages
It’s expensive
Slow penetrance
Great difficulty in counterstaining after its use
As it is easily reduced by heat, light & dust it should be stored in cool, dark place in amber glass bottle
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
Christopher Layton, John D. Bancrofti, S Kim Suvarna. Fixation of Tissues. In: Theory and practice of histological techniques by John D. Bancrofti . 8th edition.
Cullings. Histotecniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch, Stanely S. Raphael. Saunders publication 1983
