Fixative Table
Short Description
Table of Fixatives for Histopathology...
Description
Chapter 3: FIXATION AND FIXATIVES | 1
I. ALDEHYDE FIXATIVES FIXATIVE
DESCRIPTION
10% conc. is widely used made from the oxidation of methyl alcohol 37 – 40% w/v soluble in water 35%-40% gas by weight buffered to pH 7 (phosphate buffer)
PURPOSE
preserves fat and mucin preserves glycogen preserves proteins recommended recommended for nervous tissue preparation
ADVANTAGES
Fixation time: 24 HOURS
Formaldehyde (Formalin)
Precipitates may be removed by filtration or by addition of 10% methanol Bleaching of tissues may be prevented by changing the fluid fixative every three months
cheap, readily available, easy to prepare, relatively stable compatible with many stains does not overharden tissues penetrates tissues well does not precipitate proteins does not make tissues brittle colored tissue photography allows frozen tissue sections to be prepared easily DOESN’T REQUIRE WASHING OUT Used for mailing specimens
DISADVANTAGES
Fumes are irritating to nose and eyes Solution is irritating to skin (causes allergic dermatitis on prolonged contact) May produce considerable shrinking of tissues Does not harden some cytoplasmic structures adequately If unbuffered: formalin reduces both basophilic and eosinophilic staining of cells (acidity of formic acid may be used when applying the silver impregnation technique of staining) forms abundant brown pigment granules on blood containing tissues due to blackening of hemoglobin Prolonged fixation: bleaching of specimen/loss of natural colors dispersing of fat from the tissues into the fluid dissolution or loss of glycogen , biurate of sodium crystals and uric acid
10% Formol-Saline
Simple microanatomical fixative Made up of saturated formaldehyde (40% w/v)
Recommended for fixation of central nervous tissues & general postmortem tissues for histochemical examinations
Fixation time: 24 HOURS at 35˚C (95˚F) 48 HOURS at 20- 25˚C (65-77˚F)
10% Neutral Buffered Formalin/ PhosphateBuffered Formalin
most common fixative
Formula: 3.5mg Sodium dihydrogen phosphate (anhydrous) 6.5mg Disodium hydrogen phosphate (anhydrous) 100ml 40% Formaldehyde 900ml Distilled Water
recommended for preservation and storage of surgical, post-mortem and research specimen
penetrates and fixes tissues evenly preserves microanatomic and cytologic details with minimum shrinkage and distortion large specimens may be fixed for a long time provided that the solution is changed every three months preserves enzymes and nucleoproteins demonstrates fats and mucin does not overharden tissues; facilitates dissection of specimen ideal for most staining techniques (including SILVER IMPREGNATION) allows natural color of tissue to be restored upon immersion in 70% alcohol prevents precipitation of acid formalin pigments on post-mortem tissue best fixative for tissues containing iron pigments and for elastic fibers which do not stain well after Susa, Zenker or Chromate fixation requires no post-treatment after fixation, so the tissue goes directly to 80% alcohol for processing
Chapter 3: FIXATION AND FIXATIVES | 2 Similar to formalin with the following additions: slow fixative (≥ 24 hours) tissues tend to shrink during alcohol dehydration ( this may be reduced by secondary fixation ) Metachromatic reaction of amyloid is reduced Acid dye stains less brightly than when fixed with mercuric chloride
longer to prepare; time-consuming positivity of mucin to Periodic acid-Schiff (PAS) stain 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 , especially neutral fats and phospholipids
Fixation Time: 4 – 24 HOURS
Fixation time: 3 – 24 HOURS
Formal-Corrosive (Formal Sublimate)
formol-mercuric chloride solution is recommended for routine postmortem tissues
penetrates small pieces of tissue 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 cytologic structures and blood cells are well preserved no need for “washing out”; tissues can be transferred directly from fixative to alcohol
penetration is slow ( tissue sections should not be more than 1 cm thick) forms mercuric chloride deposits does not allow frozen tissue sections to be made inhibits the determination of the extent of tissue decalcification
Chapter 3: FIXATION AND FIXATIVES | 3
Alcoholic Formalin (Gendre’s) Fixative
Glutaraldehyde
post-fixation with phenolformalin for 6 hours or more can enhance immunoperoxidase studies on the tissues and in some cases, electron microscopy, if it is necessary at a later time to establish a diagnosis
made up of two formaldehyderesidues, linked by three carbon chains acts similarly to formaldehyde sometimes used for routine light microscopy buffered glutaraldehyde, followed by secondary fixation in osmium tetroxide is satisfactory for electron microscopy
used to fix sputum since it coagulates mucus
fixes lipids; especially neutral fats and phospholipids fixation is faster (reduced to one-half) can be used for rapid diagnosis since it fixes and dehydrates at the same time (i.e. frozen section) good from preserving glycogen and for microincineration technique
2.5% solution – small tissue fragments and needle biopsies
Fixation time: 2 – 4 HOURS
4% solution – large tissues less than 4mm thick
Fixation time: 6-8 HRS up to 24 HRS
more stable effects on tissues, giving a firmer texture with better tissue sections; especially of nervous tissues preserves plasma proteins better produces less tissue shrinkage preserves cellular structures recommended for enzyme histochemistry and electron microscopy more pleasant and less irritating to the nose does not cause dermatitis
produces gross hardening of tissues causes partial lysis of RBCs preservation of iron-containing pigments is poor formaldehyde foes not give as good a morphological picture as glutaraldehyde it causes little cross-linking under usual fixation conditions where low concentration of proteins are used ( glutaraldehyde is more effective at cross-linking )
more expensive less stable penetrates tissues more slowly tends to make tissue (i.e. renal biopsy) more brittle reduces PAS positivity of reactive mucin (may be prevented by immersing glutaraldehyde-fixed tissues in a mixture of concentrated glacial acetic acid and aniline oil) PRECAUTIONS: specimen vial must be kept refrigerated during fixation process swirling the vials ensures that the specimen is in contact with the fresh solution at all times as it might change
Fixation time: varies from ½ hour (for small specimens) to 2 hours (maximum contribution)
II. METALLIC fixatives A. Mercuric chloride FIXATIVE
DESCRIPTION
Mercuric Chloride
most common metallic fixative
PURPOSE
recommended for renal tissues, fibrin, connective tissues & muscles
ADVANTAGES
penetrates & hardens tissues rapidly and well nuclear components are shown in fine detail
DISADVANTAGES
penetrates poorly & causes marked shrinkage of cells, so it’s usually combined with other
frequently used in sat. aq. solutions of 5-7% widely used as a secondary fixative, reacting with a number of amino acid residues & accompanied by spectroscopic changes, probably due to reactions with histidine residues compound solutions must always be freshly prepared use of metallic forceps and metallic caps should be avoided contact of fixative with personal jewelries should be avoided
precipitates all proteins great affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining Trichome staining is excellent routine fixative of choice for preservation of cell detail in tissue photography permits brilliant metachromatic staining of cells
Zenker’s Fluid
made up of mercuric chloride stock solution to which glacial acetic acid is added to it just before use to prevent turbidity & formation of dark precipitate solutions must always be freshly prepared tissues should be cut thin (2-3mm) & hollow organs should be opened to promote complete penetration & fixation
good general preservative for all kinds of tissues gives excellent staining results recommended for fixing small pieces of liver, spleen, connective tissue fibers & nuclei
produces fairly rapid and even fixation stock solutions keep well without disintegration recommended for Trichrome staining permits brilliant staining for nuclear & connective tissue fibers compatible with most stains may act as a MORDANT to make certain special staining reactions possible
Chapter 3: FIXATION AND FIXATIVES | 4 fixative agents ( this may be counteracted by addition of acid) rapidly hardens the outer layer of the tissue with incomplete fixation of the center ( thin sections should be made ) penetration beyond the first 2-3mm is slow (not more than 5mm thickness of tissues should be used) if left in the 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 tissue difficult causes considerable lysis of RBCs and removes iron from hemosiderin INERT TO FATS AND LIPIDS leads to the formation of black granular deposits in the tissues ( may be removed by adding sat. iodine solution in 90% alcohol; iodine will be decolorized with absolute alcohol in the subsequent stages of dehydration ) reduces the amount of demonstrable glycogen compound solutions deteriorate rapidly upon addition of glacial acetic acid to formalin extremely corrosive to metals penetration is poor not stable after addition of acetic acid prolonged fixation (for more than 24 hours (will make tissues brittle and hard causes lysis of RBCs & removes iron hemosiderin does not permit cutting of frozen sections tendency to form mercuric pigment deposits or precipitates ( may be removed by dezenkerization) Bring slides to water Immerse in Lugol’s iodine (5 m in.) Wash in running water (5 min.) Immerse in sodium thiosulfate 5% (5 min.)
Fixation time: 12 – 24 HOURS
Fixation time: 12 – 24 HOURS
excellent microanatomic fixative for PITUITARY GLAND, bone marrow, spleen & liver
Zenker-formol (Helly’s solution)
Heidenhain’s Susa Solution
excellent cytologic fixative after using this fixative, the tissue should be transferred directly to a high-grade alcohol (i.e. 96% or absolute alcohol) to avoid undue swelling of tissues caused by treatment with low-grade alcohol
recommended mainly for TUMOR biopsies (especially of the skin)
Fixation time: 3 – 12 HOURS
B-5 Fixative
prior to use, add 1cc. of formaldehyde (40%) for 10cc. of B-5
Chapter 3: FIXATION AND FIXATIVES | 5 Wash in running water (5 min.) Proceed with required water-soluble stain tissue must be washed in running water for several hours (or overnight) before processing insufficient washing may inhibit or interfere with good cellular staining similar to Zenker’s brown pigments are produced if tissues (especially blood containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis ( may be removed by immersing the tissue in sat. alcoholic picric acid or sodium hydroxide)
used for BONE MARROW BIOPSIES
penetrates and fixes tissues well nuclear fixation & staining is better than Zenker’s preserve cytoplasmic granules well
penetrates and fixes tissues rapidly & evenly produces minimum shrinkage and hardening of tissues due to the 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 & 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 good fixative for cytology of bone marrow biopsies
Fixation time: 1 ½ HR – 2 HRS
prolonged fixation of thick materials may produce considerable shrinkage, hardening and bleeding ( tissues should not be >1mm) RBC preservation is poor some cytoplasmic granules are dissolved mercuric chloride tend to form in tissues (these may be removed by immersion of tissues in alcoholic iodine solution ) Weigert’s method of staining elastic fibers is not possible in Susa-fixed tissues
overfixation hardens the tissue and makes cutting difficult some B-5 solutions will form precipitate on standing (but this is of no consequence) may causing mercuric pigments to form on tissues ( de-zenkerization may be employed )
B. CHROMATE FIXATIVES FIXATIVE
DESCRIPTION
PURPOSE
ADVANTAGES
DISADVANTAGES
Chapter 3: FIXATION AND FIXATIVES | 6
Chromic Acid
used in 1-2% aq. solution, usually as a constituent of a compound fixative
used in a 3% aq. solution
Potassium Dichromate
Fixation time: 12 – 48
recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC & colloidcontaining tissues
precipitates all proteins adequately preserve carbohydrates
fixes but does not precipitate cytoplasmic structures preserves lipids preserves mitochondria (if used in pH 4.5 – 5.2) penetrates tissues well hardens tissues better and more rapidly than Orth’s fluid
Redard’s (Muller’s Fluid)
Orth’s Fluid
• strong formaldehyde (40%) is added just before use
demonstrates RICKETTSIAE & other bacteria
Fixation time: 36 – 72 HOURS
recommended for the study of degenerative processes & tissue necrosis preserves myelin better than buffered formalin
it’s a strong oxidizing agent ( a strong reducing agent, such as formaldehyde, must be added to chrome-staining fixatives before use in order to prevent counteracting effects & consequent decomposition of solution upon prolonged standing ) if solution becomes acidified, cytoplasm, chromatin bodies & chromosomes are fixed but mitochondria is destroyed
deteriorates & darkens on standing due to acidity ( solution must always be freshly prepared ) penetration is slow ( tissues should be no thicker than 2 – 3 mm) chromate fixed tissues tend to produce precipitates od sub-oxide ( tissues should be thoroughly washed in running water prior to dehydration ) prolonged fixation blackens tissue pigments like melanin ( tissues should be washed in running water prior to dehydration) glycogen penetration is poor; generally contraindicated for carbohydrates nuclear staining is poor does not preserve fats preserved hemosiderin less than buffered formalin intensity of PAS reaction is reduced same as Regaud’s fluid
C. LEAD FIXATIVES FIXATIVE Lead Fixatives
DESCRIPTION
used in 4% aq. solution of basic lead acetate
PURPOSE
ADVANTAGES
recommended for acid mucopolysaccharides fixes connective tissue mucin
DISADVANTAGES
takes up CO 2 to from insoluble lead carbonate especially upon prolonged standing ( removed
Chapter 3: FIXATION AND FIXATIVES | 7 by filtration or adding acetic acid drop by drop to lower the pH and dissolve the residue)
III. PICRIC ACID fixatives FIXATIVE
DESCRIPTION
Normally used in sat. aq. solution (approx.. 1%) yellow dye may be removed by treatment with another acid dye or lithium carbonate
PURPOSE
excellent fixative for glycogen demonstration
ADVANTAGES
stable, penetrates tissues well & fixes small tissues rapidly yellow stain prevents small fragments from being overlooked allows brilliant staining with the trichrome method suitable for Aniline stains (Mallory’s, Heidenhain’s or Masson’s methods) precipitates all proteins
DISADVANTAGES
Picric Acid
dyes tissues yellow ( dye may be removed by treatment with another acid dye or lithium carbonate ) excessive staining may be removed by placing the tissues in 70% ethanol followed by 5% sodium thiosulfate & washed with running water causes RBC hemolysis & reduces amount of demonstrable ferric iron in tissues not suitable for frozen sections because they will crumble when cut prolonged fixation makes tissues hard, brittle & difficult to section ( not longer than 12 – 24 hours; depending on size) picrates are formed upon protein & are soluble in water ( tissues must be rendered insoluble by direct immersion in 70% alcohol) fixed tissues must never be washed in water prior to dehydration picric acid is highly explosive when dry ( keep moist with distilled water or sat. alcohol at 0.5 to 1% conc. during storage ) alters and dissolves lipids interferes with Azure eosin method of staining (tissues should be thoroughly washed with alcohol )
fc
recommended for fixation of embryos & pituitary biopsies
Fixation time: 6 – 24 HOURS
produces minimal distortion of microanatomical structures can be used for general & specific stains (shrinking effect of picric acid is balanced by the swelling effect of glacial acetic acid) excellent fixative for preserving soft & delicate structures (i.e. endometrial curettings) penetrates rapidly & evenly, causing little shrinkage yellow stain is useful when handling fragmented biopsies permits brilliant staining of tissues preferred fixative for tissues to be stained by Masson’s trichrome for collagen , elastic or connective tissue (if tissue is fixed in formalin, a pre-treatment of Bouin, as a mordant prior to trichrome stain, is recommended) preserves glycogen does not need “ washing out”
Bouin’s Solution
Chapter 3: FIXATION AND FIXATIVES | 8 penetrates large tissues poorly picrates are soluble in water ( tissues must be transferred directly from fixative to 70% alcohol ) not suitable for fixing kidney structures, lipids & mucus destroys cytoplasmic structures (i.e. mitochondria) produces RBC hemolysis & removes demonstrable ferric iron from blood pigments reduces or abolishes Feulgen reaction due to hydrolysis of nucleoproteins
better and less messy than Bouin’s solution excellent fixative for glycogen
Brasil’s Alcoholic Picroformol Fixative
GLACIAL ACETIC ACID FIXATIVE
DESCRIPTION
colorless liquid
PURPOSE
DISADVANTAGES
It fixes and precipitates nucleoproteins.
called “Glacial” Acetic Acid
other fixatives to form a compound
It precipitates chromosomes and chromatin
when undiluted because it is
solution
materials; hence, is very useful in the study of
It precipitates DNA, which is split off
nuclear components of the cell. In fact, it is an
from nucleoprotein
essential constituent of most compound
since it destroys mitochondria and Golgi
valuable for the preservation of
nuclear fixatives.
elements of cells.
a water-free (anhydrous)
acetic acid that freezes and
Acetic Acid
ADVANTAGES
Normally used in conjunction with
solidifies at about 17°C.
nuclei
It causes tissues (especially those containing collagen) to swell. This property is used in certain compound fixatives to counteract the shrinkage produced by other components (e.g. mercury).
When combined with Potassium Dichromate, the lipid-fixing property of the latter is destroyed (e.g. Zenker's fluid).
It is contraindicated for cytoplasmic fixation
Chapter 3: FIXATION AND FIXATIVES | 9
Alcohol fixatives FIXATIVE
DESCRIPTION
PURPOSE
ADVANTAGES
Methyl Alcohol 100%
Isopropyl Alcohol 95%
Ethyl Alcohol
is used at concentrations of 70-100% use of lower concentrations cause hemolysis of RBC and inadequately preserved WBC
is used for fixing touch preparations some touch preparations are air dried and not fixed, for certain special staining procedures such as Wright-Giemsa It may be used as a simple fixative. frequently incorporated into compound fixatives for better results
It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues. It fixes and dehydrates at the same time.
Preserves but does not fix glycogen. Fixes blood, tissue films and smears. Preserves nucleoproteins and nucleic acids; used for histo-chemistry, especially for enzyme studies. Fixes tissue pigments fairly well.
DISADVANTAGES
Fixation Time : 18-24 HOURS
Carnoy’s Fluid
Rapid in action Tissues fized in Carnoy’s for 1 hr can be transferred directly to absolute alcohol, or an alcohol-chloroform mixture (1:1)
Recommended for fixing chromosomes, lymph glands & urgent biopsies May be used for urgent biopsy spx for paraffin processing w/in 5 hrs Used to fix brain tissues for diagnosis of rabies
Fixation Time : 1-3 HOURS
Newcomer’s Fluid
Fixation Time : 12-18 HOURS @ 3°C
Most rapid fixative; may be used for urgent biopsy specimens for paraffin processing w/in 5 hrs 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, tissues may be transferred directly to absolute alcohol, thereby shortening processing time. Rec for fixing mucopolysaccharides and nuclear proteins. Produces better reaction in Feulgen stain than Carnoy's fluid.
Penetration is slow. If left in fixative for more than 48 hours, tissues may be over hardened and difficult to cut.
Causes polarization of glycogen granules Produces considerable hardening and shrinkage of tissues Hemosiderin preservation is less than in buffered formaldehyde A strong reducing agent; should not be mixed w/ strong oxidizing agents such as chromic acid, potassium dichromate& osmium tetroxide Produces RBC hemolysis Causes considerable tissue shrinkage Suitable ONLY for small pieces of tissues due to slow penetration Harden tissues excessively & distorts tissue morphology Dissolves fat, lipids, myelin Leads to Polarization (unless very cold temp [70C] are used) Dissolves acid-soluble cell granules and pigments
Chapter 3: FIXATION AND FIXATIVES | 10
Acts both as a nuclear and histochemical fixative.
Osmium tetroxide (osmic acid) FIXATIVE
DESCRIPTION
Most common chromiumosmium acetic acid fixative used
PURPOSE
Recommended for nuclear preparation of such sections
ADVANTAGES
Fixation Time : 24-48 HOURS Flemming’s Solution
It is an excellent fixative for nuclear structures, e.g. chromosomes. It permanently fixes fat. Relatively less amount of solution is required for fixation (less than 10 times the volume of the tissues to be fixed).
DISADVANTAGES
Flemming’s Soluton w/o acetic acid
Made up of chromic and osmic acid Removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell
Recommended for cytoplasmic structures particularly the mitochondria
Same as Flemming’s
It is a poor penetrating agent; hence, is applicable only to small pieces of tissues. The solution deteriorates rapidly and must be prepared immediately before use. Chromic-osmic acid combinations depress the staining power of hematoxylin (especially Ehrlich's hematoxylin). It has a tendency to form artifact pigments; these may be removed by washing the fixed tissue in running tap water for 24 hours before dehydration. It is very expensive. Same as Flemming’s
Trichloroacetic acid FIXATIVE
DESCRIPTION
PURPOSE
Tricholoracetic Acid
reagent that is used for the precipitation of proteins and nucleic acids Also used as a decalcifier and fixative in microscopy. 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 Its softening effect on dense fibrous tissues facilitates preparation of such sections.
DISADVANTAGES
Poor penetrating agent; suitable only for small pieces of tissues or bones
Acetone FIXATIVE
DESCRIPTION
PURPOSE
ADVANTAGES
DISADVANTAGES
Acetone
Used at ice cold temperature ranging from -5°C to 4°C Fixation time may vary from several minutes (for cell smears, cryostat sections) to several hours (1-24 hours for small tissue blocks).
Its use is reserved for the fixation of cryostat sections or for tissues in which enzymes have to be preserved.
It is recommended for the study of water diffusible enzymes especially phosphatases and lipases. It is used in fixing brain tissues for diagnosis of rabies. It is used as a solvent for certain metallic salts to be used in freeze substitution techniques for tissue blocks.
Chapter 3: FIXATION AND FIXATIVES | 11 Produces inevitable shrinkage and distortion Dissolves fat Preserves glycogen poorly Evaporates rapidly
Heat fixation FIXATIVE
DESCRIPTION
Involves thermal coagulation of tissue proteins for rapid diagnosis
PURPOSE
Usually employed for frozen tissue sections and preparation of bacteriologic smears
ADVANTAGES
Fixation is better Preserves nuclear and cytoplasmic detail Suitable for frozen tissues preparation
DISADVANTAGES
Produces considerable tissue shrinkage and distortion Destroys RBC Dissolves starch and glycogen
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