Clearing is required to remove alcohol from tissues and is replaced by fluid which is miscible with wax with which tissue must be impregnated
Clearing agent is necessary as the dehydrating agents are not miscible with paraffin
Clearing agent is miscible with both dehydrating agent as well as paraffin wax
Why is the process of removing alcohol called as clearing
The process is called as clearing because in addition of removal of alcohol, the tissues are rendered translucent because refractive index of clearing agents is approximately equal to that of tissue proteins.
Name different clearing agents
Benzene
Xylene
Toluene
Choloroform
Petroleum ether
Oil of wintergreen (Methyl salicylate)
Cedar wood oil
Carbon tetrachloride
Clove oil
Dioxane
Aniline oil
What are the features of good clearing agent
Good clearing agent should
Remove alcohol quickly
Clear quickly without overhardening
Should not dissolve out aniline dyes
Should not evaporate to quickly in wax bath
How much volume of clearing agent is required
More than 10 times the volume of the tissue
Which is the most commonly used cleaning agent
Xylene
What are the advantages of xylene
Advantages of xylene are
It is cheap and rapid in action
Can be used in most of tissues
Can be used for both paraffin and celloidin embedding
What are the disadvantages of xylene
It makes tissues excessively hard and brittle, if the tissue is left for more than 3 hours in xylene
It is not suitable for brain and lymphnodes
Should be handled with care as it causes dermatitis
How do we know whether dehydration is complete or not
Pour the xylene on the tissue section. If the dehydration is not complete then the xylene becomes milky. Then the tissue should be placed again in absolute alcohol
Name the reagent used for clearing in tissues for electron microscopy
Toluene
What are advantages and disadvantages of toluene as clearing agent
Toluene causes less hardening than xylene but the clearing is much slower than xylene
What are the advantages of chloroform
It is excellent clearing agent for nervous tissue, lymph nodes and embryos as it causes little shrinkage
It does not harden tissue excessively
It is best clearing agent for large blocks
Not inflammable
What are the disadvantages of choloroform
It is expensive and evaporates rapidly from wax both
What are disadvantages of benzene
Benzene is toxic and highly flammable
What are the disadvantages of cedar wood oil
Wax impregnation after cedar wood oil is slow and 3 changes are essential
Which clearing agent used for making embryos, plants and insects transparent so that their internal structures can be seen
Oil of Wintergreen (Methyl salicylate)
What is the procedure of clearing
After dehydration of tissue, it is immersed in 2 or 3 changes of xylene
At the end of clearing tissue should look transparent.
Reference
Cullings. Histotecniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch, Stanely S. Raphael. Saunders publication 1983