PRINCIPLE AND TYPES OF

CELL FIXATION AND STAINING

Prof Varsha Baweja

Department of Zoology
Deshbandhu College
University of Delhi
vbaweja@db.du.ac.in
Fixation
• Fixation brings about the death of the cell in such a way that the structure of
the living cell is preserved with a minimum of artefacts.
• Fixation is also important in maintaining the chemical composition of the
cell.
Fixatives
• Prevent autolysis and putrefaction by rapid coagulation of cytoplasm
• Fixation results in the separation of the solid phase of the colloid from the liquid
phase. A good fixative forms ultramicroscopic precipitates which does not change
the appearance of the cell.
• It should not shrink or swell or destroy the structure of tissues or cells or makes
cells insensitive to hypotonic or hypertonic solutions
• It should rapidly and evenly penetrate the tissue
• It should give good optical differentiation
• It must harden the tissue and render it for each manipulation.
• It should render the material receptive to stains
Fixatives

• Fixing agents produce cross linkages between protein molecules. For

ex, aldehydes react with the amino, carboxyl, and indole groups of a
protein and then produce methylene bridges with other protein
molecules
+ HCHO -----NH.CH2OH
Amino Group Formaldehyde Methylol

------NH.CH2OH + ---NH-CH2-HN + H2O

Methylol Amino Group Methylene bridge
Ideal Fixatives

• It should be cheap and easily available.

• It should be stable and safe to handle.
• It should be rapid in action.
• It should cause minimal loss of tissue.
• It should not bind to the reactive groups in the tissue which are meant
for dyes.
• It should give even penetration.
• It should retain normal colour of the tissue.
• It should not impart its own colour to the tissue.
Some Common Fixatives
• Acetic acid
• Chromium trioxide
• Ethanol
• Formaldehyde
• Mercuric chloride
• Osmium tetraoxide
• Potassium dichromate
• Picric acid
Classification of Fixatives
On the basis of
• composition:
• Simple fixative – Ex: Acetic acid, Formaldehyde
• Mixture/ Compound fixatives – Ex: Bouin, Carnoy
• reaction with soluble proteins:
• Coagulant fixatives- Fixatives bring about crosslinking of proteins which
produces denaturation or coagulation of proteins so that the semifluid state is
converted into semisolid state; so that it maintains everything in vivo in
relation to each other. Thus, semisolid state facilitates easy manipulation of
tissue. Ex: Ethanol, Mercuric chloride, Chromium trioxide
• Non-coagulant fixatives- Ex: Formaldehyde, Osmium Tetraoxide, Potassium
dichromate, Acetic acid
Classification of Fixatives
On the basis of
• their association with cell proteins:
• Additive fixatives- When fixatives combine with cellular proteins, they are called as additive
fixatives. Ex: Formaldehyde
• Non-additive fixatives- when the fixative does not combine with the cellular protein but denature
them, it is called non-additive fixative. Ex: Ethanol, Mercuric chloride, Chromium trioxide
• their chemical nature:
• Aldehydes- Ex: Formaldehyde, Gluteraldehyde, Acrolein, Glyoxal
• Oxidizing agents- Fixation of lipids, Ex: Osmium tetraoxide, Potassium permanganate, Potassium
dichromate
• Protein-denaturing agents- Ex: Acetic acid, Methyl alcohol, Ethyl alcohol
• Cross-linking agents- Ex: Carbodiimide, Dimethyl suberimidate
• Unknown mechanism- Ex: Mercuric chloride (it is known to react with amines, amides, amino acids
and sulfhydryl groups, the latter being prominent in its reaction with cysteine where it is thought to
produce cross-links. It is a powerful protein coagulant which leaves tissue in a state which produces
strong staining with acid dyes. ), Picric acid (It penetrates well and fixes rapidly. It precipitates
proteins and combines with them to form picrates some of the picrates are water-soluble so must be
treated with alcohol
Commonly used fixatives for cell study
• Acetic acid and Ethyl alcohol- Chromosomes:
• Acetic acid- for swelling and softening
• Ethyl alcohol- for shrinkage and hardening
• It is rapid acting, gives good nuclear preservation, and retains glycogen. Nissl’s granules
and glycogen are preserved. It lyses erythrocytes and dissolves lipids and can produce
excessive hardening and shrinkage. Chemical composition is: Absolute alcohol = 60ml
Chloroform = 30ml Glacial acetic acid = 10 ml
• Osmium tetraoxide and Formaldehyde- Golgi complex
• Formaldehyde- Mitochondria
• Formaldehyde, Potassium dichromate or Osmium tetra oxide- Distribution of fat
droplets
• Acetone and Formalin- Enzymatic studies
Actions of commonly used fixatives
• Ethanol:
• Dissolves lipids and sets them free from lipoprotein
• Precipitates nucleic acids
• Shrinkage and hardening of tissues
• Glacial acetic acid
• Precipitates nucleoproteins
• Swells and soften the tissues
Actions of commonly used fixatives
• Formaldehyde
• Hardens the material without
shrinkage
• It forms cross links with proteins by
forming methylene bridges (-Nh-
CH2-NH)
• It reacts with primary amines to form
Schiff bases, with amides to form
hydroxymethyl compounds.
Hydroxymethyl groups condense
with another amide moiety to form
methyl diamides
Actions of commonly used fixatives
• Osmium tetraoxide:
• 0.5 – 2% solution of osmium tetraoxide is used for fixing cytoplasm,
Golgi complex, mitochondria and fat.
• It functions as a secondary fixative by reacting with lipids. It is
believed that the unsaturated bonds of fatty acids are oxidized by
OSO4 and it is reduced to a black metallic osmium (mw-254.2) which
is electron dense and adds contrast to biological tissues (secondary
stain)
• It is extensively used for electron microscopy.
Fixation Artefacts
• Extrinsic: Formed by fixative and its deposition in the tissue. For ex:
mercuric chloride fixation leads to the formation of black granules.
• Intrinsic: Formed by tissue itself during fixation. The cells may shrink,
coagulum may form, or distortion of cell may take place.
Freezing fixation
• Quick fixation and is good for enzymes and cytochemical studies.
• Freeze-drying: Freezing of tissue is followed by drying in vacuum at a
low temperature
• Freeze substitution: Tissue is transferred to acetone or alcohol after
freezing, that replaces ice crystals in the tissue.
• Heat Fixation: Heat denatures the proteolytic enzymes and prevents
autolysis. Ex: bacteria, archaea
Staining
• Chemical groups of Stains:
• Chromophoric: is the part of the molecule that is responsible for the colour of
that molecule. Ex: carboxyl group (-COOH), azo(-N = N-), nitro group (-
NO2), etc
• Auxochromic: it provides the stain an ability to attach to the tissue. An
auxochrome can increase the colour of any organic compound. E.g. benzene is
a colourless compound, but nitrobenzene is a yellow-coloured compound
(nitrobenzene contains a nitro group attached to benzene). Here, the nitro
group is a chromophore for benzene molecule.
Types of Stains
• Acidic: The chromophoric group of acidic stain is anionic (acidic) and
stains in acidic medium. Stains the cytoplasmic components, Ex:
picric acid, acid fuchsin, methyl blue, eosin, orange G, Congo red, etc
• Basic: The chromophoric group of basic stain is cationic (Basic) and
stains in alkaline medium. Ex: Crystal violet, methyl green, methylene
blue, safranine, basic fuchsin. Basic stains react with acidic substances
like nucleus, chromosomes, etc.
• Neutral: Combined properties of acidic and basic stains. Ex: Janus
green B,
Mordant
• It facilitates the adsorption of the stain to the cell component for better
staining.
• It is a chemical that fixes a dye in or on a substance by combining with
the dye to form an insoluble compound.
• The chelate formed from a mordant dye and a metal is called a lake
• The mordant is usually a double salt of potassium or ammonium with
aluminum or ferric sulphate.
• Metachromasia is a characteristic color change which certain aniline dyes
exhibit when bound to particular substances or when concentrated in
solution. For example, the basic dye toluidine blue becomes distinctly pink
when bound to cartilage matrix.
• This change in color may not be observed with normal tissues.
• Vital Stain: Stains used in the study of live cell.
• Progressive Staining
• Regressive Staining
• Counter Staining
• Dehydration
• Differentiation
• Clearing
• Mountant
• Mounting