ALDEHYDE

Formaldehyde
-

Advantage

Disadvant
age

Precautio
n

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 Hellys fluid (4-6 hours)
or Formol-sublimate (4-16 hours)

10% Neutral buffered

formalin/ Phosphate
buffered formalin

10% Formol saline

-

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

Preservation and storage of

surgical, post-mortem, and
research specimen

Best fixative for tissues

containing iron pigments and
elastic fibers
No post treatment

Metachromatic reaction of amyloid is

reduced

Positivity of mucin to PAS is

reduced
Reactivity of myelin to Weigerts
iron hematoxylin is reduced
Inert towards lipids

ALDEHYDE
Formal-corrosive/ Formalsublimate

Alcoholic formalin/ Gendres

-

Advantage

Disadvant
age

Routine post mortem tissues

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

Enhance immunoperoxidase study by

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

Lysis of RBC
Causes little cross-linking

Glutaraldehyde
-

Made up of two formaldehyde

residues

Central nervous tissue

Enzyme histochemistry and
electron microscopy

Positivity of mucin to PAS is

reduced

GLACIAL ACETIC ACID

Advantage
Disadvant
age

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

Contraindicated for cytoplasmic fixation

ACETONE
-

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

Advantage
Disadvant
age

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

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/ Hellys

solution

Zenkers Fluid
-

Advantage

Disadvant
age
Precautio
n

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

Microanatomic fixative for pituitary

gland and blood containing organs
Preserves cytoplasmic granules
Similar to Zenkers Fluid

Heidenhains Susa Solution

-

Tumor biopsy especially the skin

Cytologic fixative

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

to mercuric iodide, removed by

sodium thiosulfate

METALLIC FIXATIVES
Mercuric Chloride

Chromate Fixatives

B-5 Fixative
-

Advantage

Chromic Acid
-

Bone marrow biopsy

Cytology of bone marrow biopsy

1-2% aqueous solution

Strong oxidizing agent

Preserves carbohydrates
Precipitates proteins

Potassium dichromate
-

3% aqueous solution

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

METALLIC FIXATIVES
Chromate Fixatives
Regards/ Mullers fluid

Advantage

Disadvant
age

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

Orths Fluid
-

Early degenerative processes and

tissue necrosis
Rickettsiae and other bacteria
Myelin

Same in Regauds Fluid

4% aqueous solution of basic lead

acetate

Acid mucopolysaccharide
Fixes connective tissue mucin

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

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

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

Bouins Solution
-

Advantage

Disadvant
age

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 Massons
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

Brasils alcoholic picroformol fixative

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

Poor penetrating agents

Advantage

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

Excellent for fixing dry, and wet

smears, blood smears, and bone
marrow tissues

Isopropyl alcohol 95%

Ethyl alcohol
-

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
Carnoys fluid

Advantage

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

Newcomers fluid

Fixing mucopolysaccharides and nuclear proteins

Better reaction in Fuelgen stain
Nuclear and histochemical fixative

Disadvant
age

Dissolves fats, lipids, and myelin

Dissolves acid-soluble cell granules and pigments

OSMIUM RETROXIDE (OSMIC ACID)

Advantage

Disadvant
age
Precaution

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

Flemmings Solution

Flemmings Solution without acetic acid

-

Made up of only chromic acid and osmic acid

Cytoplasmic structures
Removal of acetic acid improve cytoplasmic details

Most common chrome-osmium acetic acid fixative used

Advantage

Permanently fixes fat

Excellent fixative for nuclear structures

Same as Flemmings Solution

Disadvant
age

Tendency to form artefact pigments

Same as Flemmings Solution

Thermal coagulation of tissue proteins for rapid diagnosis

Frozen tissue sections

HEAT FIXATION

Advantage
Disadvant
age

Preparation of bacteriologic smears

Preserves nuclear and cytoplasmic detail

Destroys RBC
Dissolves glycogen