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ACTIVITY 9
CLEARING
Clearing (de-alcoholization) is the process whereby alcohol or a dehydrating agent is removed
from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be
impregnated (e.g. paraffin) or the medium on which the tissue is to be mounted (e.g. Canada balsam).
When the dehydrating agent has been entirely replaced by the solvent, the tissue has a translucent
appearance; hence, the use of the term “clearing agent”.
Few dehydrating agents are miscible with paraffin wax. When used after alcohol dehydration,
the clearing agent serves to mix with alcohol and removes it from the tissue. It should also be miscible
with paraffin in order to facilitate the penetration of this embedding medium. The most commonly
used clearing agents for de-alcoholization in the embedding process are xylene, dioxane, chloroform,
and cedarwood.
When used after the tissue section has been stained, the clearing agent will make microscopic
tissue preparations transparent due to their high index of refraction. Aside from removing alcohol, a
clearing agent must also be miscible with Canada balsam and other resins that are used for mounting
sections. The most commonly used clearing agent for this purpose is xylene.
Glycerin and gum syrup are used when the tissue is to be cleared directly from water, as in a
frozen section. No de-alcoholization is involved in the process. The clearing agents merely improve
the refractive index of the tissue.
Because of the high refractive indices of most reagents used for de-alcoholization, tissues,
particularly embryos and parasites become transparent so that the internal structures become visible
to the naked eye. Due to this property of making tissues transparent, solutions utilized for alcohol
removal are also called “clearing agents”. Not all clearing agents, however, exhibits this property.
Most clearing agents are flammable liquids that warrant considerable caution in their use, and
the histotechnologist should be aware of the large quantities used in routine processing, so that the
safest method of use and storage can be adopted. Clearing agents with a low boiling point are
generally more readily replaced by melted paraffin, although chloroform which has a lower boiling
point than xylene in fact takes longer than the latter to clear. Viscosity also affects the speed of
penetration of the clearing agent. Prolonged exposure to most clearing agents causes the tissue to
become brittle and therefore more difficult to cut.
Questions for research:
1. What are the characteristics of a Good Clearing Agent?
2. List down the commonly used Clearing Agents and give their properties and advantages and
disadvantages in using it.