Histology Fixation

2.6 Fixation Artifacts:

Fixation is associated with a number of problems termed as fixation artifacts, e.g.

ƒ Formaldehyde fixatives give brown pigmentation to tissues.

ƒ Mercuric chloride fixatives leave a black precipitate of mercury in tissues.

ƒ Some fixatives produce shrinkage in tissues and similarly some produce swelling. 

ƒ Due to poor penetration of fixatives biochemical molecules like glycogen diffuse from

unfixed parts giving false localization or considerable loss. This is termed streaming

artifact and is mostly observed in case of glycogen.

2.7 SIMPLE FIXATIVES

2.7.1 Formaldehyde

Commercially available formaldehyde is a saturated solution of formaldehyde (HCHO) gas

in water, about 40% gas by weight. The formaldehyde available commercially is 40% but it

is considered 100% and then 10% is prepared for fixing laboratory specimens.

2.7.1.1 Advantages:

i. Formalin is cheaper, easy to prepare and stable. 

ii. It fixes the tissue in natural colour. 

iii. Shrinkage and brittleness are not caused by formalin fixatives.

iv. It is best fixative for nervous tissue. 

v. Frozen sections can be prepared with great ease from formalin fixed material.

vi. Different staining techniques can be used on tissue fixed in formalin

vii. It is a good fixative for lipids and proteins. (Mucins are well preserved)

viii. Compound fixatives of formalin can easily be prepared for special work on tissues.

ix. The blocks fixed in formalin do not require washing before processing.

2.7.1.2 Disadvantages:

i. These fixatives are toxic, irritant to skin and may cause dermatitis due to prolonged

handling.

ii. Formalin vapors are irritant to nasal mucosa and may cause sinusitis. This can be

prevented by handling the specimen in a ventilated area.

iii. It may also cause asthma in some allergic individuals.

iv. When kept for longer periods especially in the cold, formalin fixatives develop a

white encourage of paraformaldehyde which is a polymer of formaldehyde. The

polymer does not change the fixing ability of formalin and can be removed by

filtration. All commercial formaldehydes contain 11-16% methanol, which tends to

inhibit the formation of paraformaldehyde. Paraformaldehyde commonly has a

slight odor of formaldehyde due to decomposition.

v. Methanol has a denaturing effect on proteins, and this factor makes formalin

unsuitable for the fixation required in electron microscopy. However, pure

formaldehyde is suitable.

vi. Traces of formic acid are normally present in commercial formalin due to which it is

acidic. This acid can be neutralized by the addition of a small quantity of magnesium

carbonate or a few drops of sodium hydroxide (NaOH). The acidity usually reduces

the quality of staining, particularly nuclear staining.

vii. In tissues containing much blood (, e.g. spleen), unbuffered* formalin leads to

formation of dark-brown artifact pigment granules. The granules consist of acid

formaldehyde, haematin and are doubly retractile.

viii. Formalin is medium speed fixative and complete fixation requires 12-24 hours.

*formal saline + handful calcium carbonate + well shaken

2.7.2 Mercuric Chloride

It belongs to the class of fixatives of unknown mechanism. Mercuric chloride HgCl2 is a

metallic salt and included in many fixatives. At room temperature its solubility in water is

about 7% and this saturated aqueous solution is used for fixation purposes. 

2.7.2.1 Advantages:

i. These fixatives are mainly used as secondary fixatives in order to enhance the final

staining

ii. These act as a mordant due to metallic mercury, so staining reactions are excellent.

iii. These give good staining results in case of special stains, e.g. trichrome stains, which

are used for connective tissue elements and muscle fibers differentiation. Similarly

metachromatic staining for cartilage matrix and mast cell granules, and chromaffin

reaction.

2.7.2.2 Disadvantages:

i. These fixatives produce a lot of shrinkage in tissues.

ii. Fixatives containing mercuric chloride leave a black precipitate of mercury in tissues

and it must be removed. 

iii. Mercuric chloride fixatives are not stable and easily deteriorated.

iv. HgCl2 is corrosive and should not come into contact with metal surfaces. It is very

poisonous and must be handled with care.

2.7.3 Osmium Tetraoxide 

It belongs to the class of µoxidizing DJHQWV¶ fixatives. It is also known as osmic acid and is a

pale yellow powder having solubility about 6% at 20° C. Osmium tetraoxide OsO4 is

extremely volatile and is easily reduced by contact with organic matter or by exposure to

day light. It ought to in this manner be kept in a dull, synthetically clean container.

2.7.3.1 Advantages:

i. It is the only fixative that can permanently fix lipids (Golgi bodies and mitochondria

are also preserved which are cytoplasmic organelles). 

ii. It is seldom used alone as a fixative, but is usually combined with chromium salt. Its

vapors can be used for fixation.

iii. It can also be used for electron microscopy work.

2.7.3.2 Disadvantages:

i. One of its lower oxides, i.e. osmium dioxide OsO2 can become deposit in the cornea

resulting in blindness.

ii. The fixative is expensive and dangerous and penetration is also poor.

iii. Tissues fixed in OsO4 require washing out, i.e. they are set in running faucet water

for hours before taking them to reducing alcohols. If vapors are used for fixation,

washing out is not required and vapors penetration is also better than solution.

2.7.4 Picric Acid

It belongs to the class of fixatives of unknown mechanism. Its solubility is about 1% in

water. It is always kept in saturated aqueous solution form. Picric acid C6H3N3O7 is

explosive when dry, therefore for fixation purposes, yellow coloured solution is used.

2.7.4.1 Advantages:

i. It is recommended for histochemical demonstration of glycogen.

ii. Picric acid imparts a yellow colouration that acts as a merit in case of handling tiny

biopsies which have a chance of misplacing.

2.7.4.2 Disadvantages:

i. The yellow colouration is a disadvantage because it impairs final staining and so it

requires several washes in alcohol or xylene to remove yellow colour.

ii. It precipitates all proteins and combines with them to form picrates. These picrates

are solvent in water, and the tissue should not interact with water until the

picrates have been delivered insoluble by treatment with liquor.

2.7.5 Potassium Dichromate

Potassium Dichromate K2Cr2O7 is one of the oldest and is most widely used simple fixative.

Two totally various types of obsession can be delivered relying on the pH of the

solution. If pH is below 4.6, the result is similar to those produced by chromic acid. If pH is

above 4.6 the cytoplasm is homogenously preserved and the mitochondria are fixed.

2.7.5.1 Advantages and Disadvantages:

i. It is recommended for myelinated nerve fibers in which it fixes myeline lipids. This

property is lost if mixed with glacial acetic acid.

ii. Tissues fixed in K2Cr2O7 UHTXLUH³:DVKLQJRXW´ (to remove oxidizing agent).

iii. It is a metallic salt so act as a mordant and may be used as a secondary fixative.

iv. Formaldehyde fixed tissues are placed in 3% potassium dichromate for about 8

KRXUV7KLVWUHDWPHQWLVQDPHGDV³3RVW-FKURPLQJ´DQGHQKDQFHVILQDOVWDLQLQJ

v. It is prepared by dissolving crystals of the anhydride CrO3 in distilled water, and is

stored as a 2% stock solution. It is a strong oxidizing agent so not combined with

reducing agents such as alcohol and formalin. It is a strong protein precipitant, and

SUHVHUYHVFDUERK\GUDWHV7LVVXHVUHTXLUH³ZDVKLQJRXW´WRDYRLGWKHIRUPDWLRQRIWKH

insoluble sub-oxide.