Histology Fixatives     

 2.7.6 Ethanol

Ethyl alcohol C2H6O belongs to the class of fixatives of protein denaturing agents. It is a

colourless liquid that is readily miscible with water. Earlier it was used as a simple fixative

but now a day its use as a simple fixative is confined to histochemical methods. It is

frequently incorporated into compound fixatives.

2.7.6.1 Advantages and Disadvantages:

i. As a simple fixative it is used at a concentration of 70-100% which preserves

glycogen but GRHVQ¶W fix it. 

ii. It is a reducing agent and should not be mixed with chromic acid, osmium tetraoxide

and potassium dichromate.

iii. It produces a lot of hardening and shrinkage of tissues.

iv. It is also a highly flammable solution.

v. It is good for enzyme demonstration.

2.7.7 Acetic Acid

Acetic Acid CH3COOH is a colourless solution with a pungent smell. At 17°C it solidifies,

which accounts for its name glacial acetic acid. It belongs to class of protein denaturing

agents.

2.7.7.1 Advantages:

i. It is often used by cytologists in studying chromosomes.

ii. It is very useful for nuclear studies because of its chromatin-precipitating properties.

iii. It is a powerful precipitant of nucleoprotein.

iv. It destroys mitochondria, golgi bodies and also RBCs so acts as a lytic agent.

v. The lytic agent action is a merit in case of cytological smears mixed with a lot of

blood which is hiding details of other cells and so when fixed in CH3COOH the red

cells are lyzed and details of other cells become visible.

2.7.7.2 Disadvantages:

i. The lytic action is a demerit when handling bone marrow biopsy because in that case

we have to preserve RBC series. 

ii. When used alone it causes swelling so it is used in certain compound fixatives to

counteract the shrinkage produced by other components, e.g. mercuric chloride and

acetic acid.

iii. If it is mixed with K2Cr2O7, the lipid fixing ability of K2Cr2O7 is lost. 

2.7.8 Trichloro Acetic Acid

Trichloroacetic acid [CCl3COOH] is sometimes incorporated into compound fixatives. It is

a general protein precipitant but has a swelling effect on many tissues to counter the

shrinkage produced by other simple fixatives. It can be used as a slow decalcifying agent

also, the conditioning impact which it has on thick stringy tissue is found to work with the

preparation of sections from blocks of this nature.

Box-1

Important Notes:

Apart from the size and thickness of the piece of tissues to be fixed, certain other considerations are

of importance:

ƒ Tissues containing a large amount of mucus fix slowly and poorly because the mucus prevents

penetration of the fixative. Whenever possible, e.g. in the case of pseudo-mucinous cysts of the

ovary, it is advisable to remove as much of the mucus as possible by washing with normal

saline. The same procedure may be applied to tissues containing blood, or organs containing

very large amount of blood e.g. lungs.

ƒ Fatty and lipomatous tissues fix slowly and blocks of such tissues should be thin and may

require longer time than average in fixative. 

ƒ Like all chemicophysical reactions, fixation is accelerated by agitation, and this is one of the

advantages of the mechanical tissue processors.

ƒ Moderate heat 37-56°C will accelerate fixation but it also hastens and accentuates autolytic

changes in the deeper parts of the tissues block before the fixing agent gains access to these

regions.

2.8 Osmotic Considerations in Fixation:

An ideal fixative should not cause swelling or shrinkage of cells due to osmotic factors. In

practice, only a few fixatives are isotonic. In general, heavy metal fixatives and those that

act mainly as protein precipitants, e.g. picric acid causes shrinkage whatever the osmotic

pressure of their solutions is. Acetic acid 5% is markedly hypertonic in relation to body

fluids leads to swelling of tissues, especially of collagen. Formaldehyde because of its

small, freely diffusible organic molecule and polymerizing action, must be used in as nearly

isotonic solution as possible. If the solution is hypotonic, swelling will be the result.

2.9 Factors Affecting Fixation

There are number of factors which can affect the fixation process. These factors are briefly

described below:

2.9.1 pH:

Tissues are composed of different types of proteins which are fixed or precipitated at

different pH values. Generally fixation is done by using fixatives having pH range of 5-8.

However sometimes a specific pH is required for proper fixation, e.g. gastric mucosal

biopsy is properly fixed at a pH value of 5.5. Similarly for chromosomal studies, nucleus

require proper fixation so a pH of 4.6 or below is recommended because at such low pH

nuclear proteins are well precipitated. Change of pH will change number of ions so reaction

will either increase or decrease. So the increase or decrease in rate of reaction will have

detrimental effect or simply harmful effect causing problems in microscopy.

2.9.2 Temperature:

Generally rise in temperature will increase rate of fixation, e.g. we use heated formalin

(60°C) in case of 2-3mm biopsy so the reaction between fixative and proteins will enhance

but diffusion of molecules and autolysis are also enhanced with rise in temperature. The

diffusion of molecules is a disadvantage for biochemical studies and therefore for such

studies tissues are fixed at 0-4° C to avoid diffusion of the molecules. Another aspect is that

we are using various inflammable chemicals in histopathology laboratory. So heating is a

very risky process. At the end, taking conclusion of all this we can say that if there is no

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2.9.3 Penetration of Fixatives:

The penetration of fixatives is usually slow because cells are bounded by semi-permeable

membrane. The intercellular substance also resist with penetration of the fixative.

Penetration of fixatives can be shown by the following relationship with time:

d Į t

d = K t

Where K is constant of proportionality and is known as Coefficient of Diffusibility. By rearranging the equation,

K = d / t

,I ³G´ LV WDNHQ LQ PLOOLPHWHUV DQG WLPH LQ KRXUV WKHQ ³&RHIILFLHQW RI Diffusibility´ LV

defined as penetration in mm/hours.

7KHµCoefficient of Diffusibility [.@¶ is different for different fixatives, e.g.

Fixatives 9DOXHRI³.´

10 % Formalin at room temperature 0.78 mm/hour

01 % Picric acid C6H3N3O7 0.5 mm/hour

Ethanol C2H6O 01 mm/hour

03% Potassium dichromate 1.33 mm/hour

So, potassium dichromate K2Cr2O7 is highly penetrating fixative whereas picric acid

C6H3N3O7 is a slow penetrating fixative. In this way, penetration affects the fixation

process.

2.9.4 Concentration of Fixatives:

Generally, concentration will have a positive effect. By concentrating the fixative, the

fixation process will be enhanced because in that case more number of molecules will be

present. But there are a few limitations to this:

a. Firstly, some of the fixatives are very expensive and we cannot use them in

concentrated form.

b. Secondly, maximum solubility of fixatives determines their concentration at which

they should be used, e.g. 1% picric acid is used for fixation because at this

concentration a saturated solution is obtained. If we want to use it at 10%

concentration, it will not be possible because to prepare it more heat is required and

that has many adverse effects.

Sometimes a fixative can be effective even at a low concentration, e.g. 3% glutaraldehyde is

a fixative normally used at 3% concentration but it will be effective even when the

concentration is reduced to 0.25%.

2.9.5 Time of Fixation:

Different fixatives are employed for different periods of time depending upon their reaction

and penetration. When tissues biopsies are kept in different fixatives for prolonged periods,

certain physical changes like shrinkage, hardness and brittleness of tissue biopsies is

observed.

In addition to these changes, certain chemical changes are also brought about by fixatives,

e.g. prolonged preservation in oxidizing agents result in oxidative cleavage of proteins. By

this cleavage, there is a considerable loss of peptides and amino acids.

2.9.6 Osmolarity of Fixatives:

Hypertonic fixatives cause shrinkage whereas swelling is observed in case of hypotonic

fixatives. There are no such effects in case of isotonic fixatives but such fixatives are

usually slow penetrating giving poor fixation. Normally a slight hypertonic fixative is used.