Fixatives

ALDEHYDE Formaldehyde -

Advantage

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Disadvant age

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Precautio n

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Formaldehyde – gas production by methyl alcohol 10% or 5% solution Preserves fat and mucin Preserves but does not precipitate protein; allows tissue enzyme study Allows natural colors to be restored (after immersion in 70% alcohol) Used for mailing specimen; “tolerant fixative” Reduces basophilic and eosinophilic staining cells Brown pigments on blood containing tissues (removed by alcoholic picric acid or 1% KOH in 80% ROH) Bleaching of specimen Dispersal of fat to liquid Loss of glycogen Induce precipitation of paraformaldehyde Addition of methanol prevent decomposition to formic acid Concentrated solutions must never be neutralized Cadmium or cobalt are added to prevent fat dissolution Formic acid formation buffered by magnesium carbonate or calcium carbonate or calcium acetate (but leaves calcium deposit) If post fix with osmic acid, tissue must not be washed Improve staining by secondary fixation in Helly’s fluid (4-6 hours) or Formol-sublimate (4-16 hours)

10% Neutral buffered formalin/ Phosphate buffered formalin

10% Formol saline -

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Saturated formaldehyde diluted to 10% with NaCl Fixation of central nervous tissue and post-mortem tissues Preserves microanatomic and cytologic details Preserves enzymes and proteins Demonstrates fats and mucin Silver impregnation Allows natural colors to be restored

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Preservation and storage of surgical, post-mortem, and research specimen

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Best fixative for tissues containing iron pigments and elastic fibers No post treatment

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Metachromatic reaction of amyloid is reduced

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Positivity of mucin to PAS is reduced Reactivity of myelin to Weigert’s iron hematoxylin is reduced Inert towards lipids

ALDEHYDE Formal-corrosive/ Formalsublimate

Alcoholic formalin/ Gendre’s -

Advantage

Disadvant age

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Routine post mortem tissues

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Silver reticulum methods Cytological structures and blood cells are well preserved No need for “washing-out” Fixes lipids Forms mercuric chloride deposits Not allow frozen tissue section to be made Inhibit determination of extent of decalcification

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Enhance immunoperoxidase study by post-fixation with phenol formalin (6 hours) Fixes and dehydrates Preserve glycogen Microincineration technique (mineral elements from ashes) Fix sputum

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Lysis of RBC Causes little cross-linking

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Glutaraldehyde -

Made up of two formaldehyde residues

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Central nervous tissue Enzyme histochemistry and electron microscopy

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Positivity of mucin to PAS is reduced

GLACIAL ACETIC ACID Advantage Disadvant age

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Solidifies at 17 degrees Fixes and precipitates nucleoproteins Precipitates chromosomes and chromatin material; essential constituent in nuclear fixatives Causes tissue to swell

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Contraindicated for cytoplasmic fixation

ACETONE -

Used at ice cold temperature ranging from -5 degrees to 4 degrees

Advantage Disadvant age

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Study of water diffusible enzymes especially phosphatases and lipases Fixing brain tissues for diagnosis of rabies Solvent for certain metallic salts Fixative and dehydrating agent Dissolves fat Poorly preserves glycogen

METALLIC FIXATIVES Mercuric Chloride

Advantage

Disadvant age Precaution

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Most common metallic fixative Used as secondary fixative reacting with a number of amino acids residues Contain black precipitates of mercury (except Susa) Trichrome staining Immunoperoxidase techniques Nuclear components shown in fine details Precipitates protein Greater affinity to acid dyes; cytoplasmic staining Tissue photography Brilliant metachromatic staining of cells Renal tissue, Fibrin, connective tissue, and muscles Lysis of RBC; removal of iron from hemosiderin Inert to fats Reduces amount of glycogen Remove black deposits by adding saturated iodine solution in 96% alcohol Avoid use of metallic forceps or caps

Zenker formol/ Helly’s solution

Zenker’s Fluid -

Advantage

Disadvant age Precautio n

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Mercuric chloride stock solution to which glacial acetic acid has been added just before use Small pieces of spleen, liver, connective tissue, and nuclei Microanatomic fixative for pituitary gland and blood containing organs Preserves cytoplasmic granules Lyse RBC Removes iron from hemosiderin Does not permit cutting of frozen sections De-zenkerization – removal of pigments; oxidation with sodium

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Microanatomic fixative for pituitary gland and blood containing organs Preserves cytoplasmic granules Similar to Zenker’s Fluid

Heidenhain’s Susa Solution -

Tumor biopsy especially the skin Cytologic fixative

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Silver impregnation Fibrous connective tissue Transferred directly to 95% alcohol or absolute alcohol Poor RBC preservation Dissolved cytoplasmic granules Weigert’s method of staining elastic fibers not possible

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to mercuric iodide, removed by sodium thiosulfate

METALLIC FIXATIVES Mercuric Chloride

Chromate Fixatives

B-5 Fixative -

Advantage

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Chromic Acid -

Bone marrow biopsy

Cytology of bone marrow biopsy

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1-2% aqueous solution Strong oxidizing agent

Preserves carbohydrates Precipitates proteins

Potassium dichromate -

3% aqueous solution

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Precipitates cytoplasmic structures Preserves lipids Preserves mitochondria (pH 4.55.2 - mitochondria; acidic pH – cytoplasm, chromatin body, and chromosomes)

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METALLIC FIXATIVES Chromate Fixatives Regard’s/ Muller’s fluid

Advantage

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-

Disadvant age

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Chromatin, mitochondria, mitotic figures, Golgi bodies, RBC, and colloid containing tissues Produce precipitates of sub-oxide Prolonged fixation blackens tissue pigments Poor glycogen penetration Poor nuclear staining Does not preserve fats Intensity of PAS is reduced

Lead Fixatives

Orth’s Fluid -

Early degenerative processes and tissue necrosis Rickettsiae and other bacteria Myelin

Same in Regaud’s Fluid

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4% aqueous solution of basic lead acetate

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Acid mucopolysaccharide Fixes connective tissue mucin

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Takes up CO2 to form insoluble lead carbonate (removed by filtration or adding acetic acid drop by drop to lower pH)

PICRIC ACID FIXATIVES

Advantage

Disadvant age

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Dyes the tissue yellow (removed by treatment with another acid dye or lithium carbonate)

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Glycogen demonstration Yellow stain taken in by tissues prevents small fragments being overlooked Brilliant staining with trichrome method Suitable for Aniline stain Precipitates proteins Lyse RBC Not suitable for frozen sections Never be washed in water Excessive staining of tissues Highly explosive when dry Dissolves lipids Interferes with Azure eosin method of staining

Bouin’s Solution -

Advantage

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Disadvant age

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Embryos and pituitary biopsy Minimal distortion of micro-anatomical structures Excellent preservative for preserving soft and delicate structures Preferred fixative for tisues to be stained by Masson’s trichrome for collagen, elastic, or connective tissue Preserves glycogen Does not need “washing-out” Not suitable for fixing kidney structures, lipid, and mucus Destroys cytoplasmic structures Lyse RBC Reduces Fuelgen reaction due to hydrolysis of nucleoproteins

Brasil’s alcoholic picroformol fixative

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Fixative for glycogen

TRICHLOROACETIC ACID -

Fixative and decalcifying agent Precipitates proteins Swelling effect on tissues serves to counteract shrinkage Softening effect on dense fibrous tissues

Disadvant age

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Poor penetrating agents

Advantage

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Rapidly denatures and precipitates proteins Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films, and smears Photographic work using 80% alcohol Fixative and dehydrating agent Preserve glycogen Preserve nuclear stain Lower concentration will cause RBC lysis and inadequately preserve leukocytes Dissolves fats Causes glycogen granules to move towards the poles or ends of the cells (polarization)

Advantage

ALCOHOLIC FIXATIVES

Disadvant age

Methyl alcohol 100% Advantage

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Excellent for fixing dry, and wet smears, blood smears, and bone marrow tissues

Isopropyl alcohol 95%

Ethyl alcohol -

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Fixing touch preparations -

Disadvant age

Preserves nucleoproteins and nucleic acids Fixes tissue pigments Strong reducing agent; should not be mixed with chromic acid, potassium dichromate, and osmium tetroxide which are strong oxidizing agents

ALCOHOLIC FIXATIVES Carnoy’s fluid

Advantage

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Chromosomes, lymph glands, and urgent biopsy Fix brain tissue for diagnosis of rabies Most rapid fixative Permits good nuclear staining and differentiation Preserves Nissl granules and cytoplasmic granules Preserves nucleoproteins and nucleic acid

Newcomer’s fluid

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Fixing mucopolysaccharides and nuclear proteins Better reaction in Fuelgen stain Nuclear and histochemical fixative

Disadvant age

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Dissolves fats, lipids, and myelin Dissolves acid-soluble cell granules and pigments

OSMIUM RETROXIDE (OSMIC ACID)

Advantage

Disadvant age Precaution

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Pale yellow powder which dissolves in water to form a strong oxidizing solution Causes complete denaturation of proteins Fixes conjugated fats and lipids; stained black Preserves cytoplasmic structures Extensively used for neurological tissues Brilliant nuclear staining with safranin Fixes materials for ultrathin sectioning in electron microscopy Precipitates and gels proteins Uniformly granular nuclei with clear cytoplasm background Some are better fixed in vapor of osmium tetroxide, thus eliminating “washing-out” Forms black precipitate Inhibits hematoxylin Black osmic oxide crystals may be dissolved in cold water Addition of saturated aqueous mercuric chloride solution prevents formation of precipitates

Flemming’s Solution

Flemming’s Solution without acetic acid -

Made up of only chromic acid and osmic acid Cytoplasmic structures Removal of acetic acid improve cytoplasmic details

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Most common chrome-osmium acetic acid fixative used

Advantage

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Permanently fixes fat Excellent fixative for nuclear structures

Same as Flemming’s Solution

Disadvant age

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Tendency to form artefact pigments

Same as Flemming’s Solution

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Thermal coagulation of tissue proteins for rapid diagnosis Frozen tissue sections

HEAT FIXATION

Advantage Disadvant age

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Preparation of bacteriologic smears

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Preserves nuclear and cytoplasmic detail

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Destroys RBC Dissolves glycogen