Histology: IMPREGNATION OR INFILTRATION

IMPREGNATION OR INFILTRATION

 4.3.8 Carbon disulphide paraffin oil and methyl benzoate are less commonly used

clearing agents.

Box 6

In certain cases of tissues processing the clearing stage can be omitted because we can use such a

dehydrating agent which is miscible both with water and paraffin wax. Two such examples are:

a. Dioxane (diethylene dioxide)

b. Cellosolve (ethylene glycol monoethyl ether)

These two dehydrating agents are miscible with paraffin wax and so it is not necessary to use any

clearing agent. However these two chemicals are very costly, slow penetrating and their vapours

are toxic. Therefore, the tissue processing involving clearing step is preferred. On the other hand,

advantage of dioxane and cellosolve is that these reagents can be used again and again for

dehydration purpose. These can themselves be dehydrated by CaCl2 and calcium oxide or

quicklime (CaO).

4.4 IMPREGNATION OR INFILTRATION

After clearing, tissue biopsies are transferred to some suitable solidifying medium which

replaces the clearing agent and then completely fills the natural cavities, spaces and

interstices of the tissues. Later on this medium is solidified and biopsies are internally

supported. An impregnating media must enter the tissue spaces in liquid form and then

solidify to support the tissues or cells. Apart from giving support, handling of tiny biopsies

is also facilitated by the step of impregnation and then by embedding.

Impregnating Media:

A wide range of impregnating media is available. Some of which are listed below:

i. Paraffin Wax
ii. Paraplast
iii. Celloidin
iv. L.V.N
v. Carbowax

Generally speaking, the volume of the impregnating medium should be at least 25 times the

volume of tissue.

4.4.1 Paraffin Wax

It is the most widely used impregnating medium obtained by the cracking of mineral oil and

is a mixture of saturated hydrocarbons. Various varieties of paraffin wax having melting

point (MP) in the range of 40 ± 70o C are available which can be sued according to the

32

temperature of laboratory. Normally a paraffin wax of melting point (MP) 54 ± 58o C is

used.

Nowadays different additives are added by the manufacturer, to get a proper MP and

moreover to increase the stickiness of the wax. Lard, beeswax, satiric acid, rubber and

ceresin are various examples of the additive used. Histopathological handling of tissues

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Advantages:

i. Paraffin wax is cheaper, free of colouration and non-reactive to tissue

components.

ii. Paraffin wax sectioning techniques require minimal supervision during the

embedding and infiltration stages.

iii. A large number of specimens can be processed by this technique because it is easier

in handling of specimens.

iv. Paraffin wax blocks and sections can be stored at room temperature.

v. Paraffin wax sections can be transported without any special treatment.

vi. Very thin sections can be obtained from paraffin wax blocks.

vii.A multitude of staining techniques can be applied, after paraffin wax impregnation

and embedding.

viii. Serial sections can also be easily produced by this technique.

ix. Paraffin wax is suitable for tissue blocks of different consistency, i.e. soft as well as

tough blocks can easily be support by paraffin wax.

Disadvantages:

i. The most important disadvantage is that heat is involved in melting of paraffin wax

and sometimes overheating results in causing a lot of harness and brittleness to

tissues.

ii. Paraffin wax undergoes shrinkage on cooling due to which cells in tissues may

disrupt. Normally 10% shrinkage is caused on paraffin wax cooling.

iii. Sometimes very tough tissue material is received in the laboratory that can only be

supported by celloidin not by paraffin wax.

4.4.2 Paraplast:

Paraplast is a mixture of purified paraffin wax and several synthetic plastic polymers. Its

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paraffin wax. In case of paraplast sudden cooling is not required during embedding and

while sectioning it is not necessary to keep the blocks on ice cubes. The blocks obtained are

more uniform and it is more resilient than paraffin wax and allows the sectioning of large

blocks and dense bone blocks with great ease.

4.4.3 Carbowax

Higher alcohols, e.g. those with 12 or more carbon atoms are solid at ordinary room

temperatures. Those with 18 ± 22 carbon atoms in their molecules have physical

characteristics that are suitable for tissue embedding. Such alcohols are polyethylene glycol

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For routine tissues, four changes of carbowax, i.e. 70%, 90% and two times in 100% at the

56 o C are used. The length of time is 30 minutes, 45 minutes and 1 hour respectively.

Carbowax is soluble and miscible with water, so from aqueous fixative, tissues can directly

be taken to carbowax and there is no need of dehydration and clearing stages. It does not

remove substances like lipids and neutral fats, thus allowing these substances to be

demonstrated in thin sections. This technique is also good for many enzyme histochemical

studies. Moreover, it reduces shrinkage and distortion.

Disadvantages:

i. The blocks of carbowax must not be allowed to come into contact with water or ice,

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ii. During the use of carbowax, blocks and unstained sections must be stored in dry

airtight containers coated with paraffin wax in a cool atmosphere. Otherwise

moisture at room temperature softens the blocks.

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purpose special mixtures are used, e.g. Pearse

Pearse: 

Diethylene glycol 40 parts

Distilled water 50 parts

Formalin 40% 10 parts

Blank and McCarthy:

0.02% gelatin equal parts

0.02% potassium dichromate equal parts

Boil for 5 minutes, cool and filter.

 4.4.4 Celloidin

Celloidin is the trade name given name to a purified form of nitro-cellulose. It is available

in an amorphous yellowish wood, like substance dampened with alcohol. The working

solution is prepared in equal parts of diethyl ether and ethyl alcohol (solvents). The strength

of working solutions are 2, 4 and 8%.

During embedding, solidification of celloidin occurs by simple evaporation process or can

be hastened by dipping the blocks in chloroform. It is very good for neuropathological

biopsies. It is of particular value as a histological embedding medium for sectioning hard

tissues of a mixed consistency. Moreover sectioning of very thick sections is also facilitated

by celloidin.

Disadvantages:

i. By using celloidin, thin and serial sections are difficult to obtain and only thick

section of 5 ± 10 u can be produced. 

ii. Evaporation is a constant problem when using celloidin and the working solutions

ought to continuously be put away in bottles fitted with ground-glass plugs.

iii. It must be remembered that one of the solvent, i.e. ether is dangerous because of its

vapours and therefore celloidin should never be used in the vicinity of open flame.

4.4.5 L.V.N. 

Low Viscosity Nitrocellulose is similar to celloidin but due to low viscosity, thinner

sections are easily produced. Moreover a harder block is formed with L.V.N than with

celloidin. The L.V.N sections have a tendency to crack, but plasticizers can be incorporated

into the medium to overcome this problem.