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Dehydration, Clearing & Wax Impregnation. Dr.Samah Kotb Nasr Eldeen. Dehydration. Dehydration:-. Is the first stage in processing after fixation. Is the removal of water and some of the lipid tissue fluids. This is done by many compounds.

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  • Is the first stage in processing after fixation. Is the removal of water and some of the lipid tissue fluids. This is done by many compounds.

Most of these compounds are alcohols. Some of these are hydrophilic and remove water from the tissues; but others dehydrate by repeated dilution of the aqueous tissue fluid.

dehydrating substances
Dehydrating Substances
  • ALCOHOLS: as Ethyl, Methyl & Butyl. Ascending grades of alcohols are used. The lower strength used depends on the type of tissue (starting with the lower strength when the tissue is soft). For routine surgical tissue 70% is accepted to be the lowest strength.

Anhydrous Copper Sulphate (ACS) or called white copper sulphate is added to the last dehydrated bath to:

1. Check the presence of water when it changes to blue.

2. Elongate the life of the alcohol.


Acetone: it is used when time is important i.e to speed up dehydration. No dilutions. Generally, it is used for urgent manual processing.


3) Dioxine: (di ethylene dioxide) also used to speed up dehydration. Sometimes used for manual processing but it has many disadvantages:

  • It is toxic.
  • Needs good ventilation.
  • Is the removal of the dehydrating agent. Most of the clearing agents have similar refractive index of protein and they make the tissue translucent. This indicates that the clearing is complete.

The selection of the clearing agent depends on the:

  • The speed of the removal of the dehydrating agent.
  • To be removed easily by the impregnating medium.
  • Harmless to the tissue.
  • Flammability.
  • Toxicity.
  • Cost.

Most of the clearing agents are flammable. Generally those of the low boiling point are rapidly removed by paraffin wax more than those of higher boiling point.


The removal of clearing agents by paraffin wax is enhanced under vacuum (not complete vacuum) because this will affect the tissue.

clearing agents
Clearing Agents
  • According to the properties and effects; they are divided into 3 groups:
  • Group I: Xylene, Toluene & Benzene.
  • Group II: Chloroform.
  • Group III: Cedar wood oil.


Xylene, Toluene & Benzene

  • They are inflammable.
  • Raise the refractive index of the tissue and make it translucent.
  • The process takes about 3 – 6 hrs for 3 – 4 mm thick blocks.
  • Benzene not recommended because of its carcinogenic properties.



  • Chloroform it’s not inflammable.
  • Does not raise the refractive index; so the tissue will not be translucent.
  • Slower in the action and slower to get rid of it by paraffin wax.
  • Needs 6 – 24 hrs for 3 – 5 mm thick blocks.


Cedar wood oil

  • Raise the refractive index of the tissue.
  • Slower in the action.
  • Not used in routine lab because the cost is very high.
  • Good for research; particularly when dealing with insects or embryological work.

Beside these clearing agents mentioned before, there are many like:

  • Paraffin Oil.
  • Carbon Tetrachloride.
  • Carbon Disulphide .
wax impregnation
Wax Impregnation:-
  • Its a removal of the clearing agent. Wax is obtained from the process of oil refinery. Its of different melting points about 40º – 70º C.

Soft wax has lower melting point whereas the hard wax has a higher melting point. The temperature should not exceed 2 – 3º C the melting point of the wax.


Some substances are added to paraffin wax in different ratio to modify the wax consistency and its melting point. These include:

  • Bees’ wax.
  • Rubber.
  • Ceresin.
  • Plastic.

The purpose of the addition of these substances are:

  • Increase the hardness of the wax; in order to cut thinner sections.
  • Increase the hardness of the wax; to give the necessary support when cutting hard tissues.
  • To aid the production of the serial sections.
  • Alter the crystalline structure of paraffin wax.

There are other types of wax beside paraffin wax:

  • Polyester wax.
  • Ester wax.
  • Fibrowax.
  • Paraplast wax.




Manual: For routine and urgent cases. This takes a long time. Prolong immersion should be in a tolerant fluid. Its now taken over by automation.


Automatic processing: carried on by automatic machines. It takes less time and it has the following advantages:

  • Automatic transfer.
  • Safety device.
  • Wax detector.

Reduced pressure can be applied to speed up wax impregnation.

  • Some machines are designed to work with the fluid exposed to heat to speed up and improve processing.
  • The disadvantage is when electricity goes off (power failure).





Paraffin wax of variable melting points can be used for embedding. This depends on the type of the tissue. If the tissue is hard; high melting point wax is used whereas low melting point wax is used with soft tissues.


Certain precautions should be taken during embedding:

  • No trace of the clearing agent.
  • Wax should not contain any dust particles.
  • Blocks should be cooled to reduce wax crystal size.

To make tissue blocks; different ways are used:

  • Leuckhart “L” shaped embedding boxes.
  • Paper boats.
  • Ice trays.
  • Plastic embedding moulds.
  • Plastic embedding rings.
  • Cassette bases.

Orientation of the tissues’ blocks:-

Most blocks cut from the largest area of the tissue but there are some exceptions:

  • Tissue of tubular natures are cut transversely.
  • Skin is cut a plane at right angle to the surface.
  • Muscle biopsies are cut transversely and longitudinally; when a particular feature is present on one side of the tissue.