1. What will happen to the clearing agent if the tissue is incompletely dehydrated?

Complete dehydration should be achieved before proceeding to the next

routine processing, which is clearing or dealcoholization since most clearing agents are
not miscible with aqueous fixatives. Clearing, on the other hand, is performed to remove
alcohol or the dehydrating agent from the tissue and replace it with a clearing agent
prior to impregnation and embedding since alcohol is highly immiscible with wax.
However, if the tissue is not completely dehydrated, clearing agents such as xylene will
not act accordingly or perform its function since the tissue may still contain the aqueous
fixative and water in which the clearing agent is immiscible with. As a result of
inadequate dehydration, the tissue will not be prepared prior to impregnation resulting
in a soft, mushy tissue block.

2. What is the best clearing agent for routine work and why?

Xylene is the best clearing agent for routine work because it has the different
characteristics of a good clearing agent. It clears within 15 to 30 minutes and makes
tissues transparent. It is miscible with absolute alcohol as well as paraffin, and it does not
extract out aniline dyes. Xylene is readily replaced by wax during impregnation and
embedding since it evaporates quickly in a paraffin oven. Also, it is reasonably cost
effective and works well for short-term clearing of tissue blocks.

3. What are the factors affecting clearing of specimens?

There are several factors that affect the clearing or dealcoholization of

specimens including the clearing agent’s boiling point, viscosity, as well as prolonged
exposure to the clearing agent. Clearing agents that have low boiling point are usually
more easily replaced by melted paraffin. Viscosity, on the hand, affects the speed of
penetration of the clearing agent. In addition, prolonged exposure to most clearing
agents may lead to brittleness of the tissue, thereby making it more difficult to cut.

4. What are the precautionary/safety measures to be observed in performing paraffin

infiltration?

The following are some of precautionary/safety measures that must be taken into
consideration when performing paraffin infiltration: (1) the tissue should not be left in the
paraffin over for longer than 4 hours; (2) do not overheat the tissue to avoid hardening
and brittleness; (3) the paraffin oven should be maintained at a temperature 2- to
5-degree Celsius above the melting point of the paraffin; (4) paraffin wax should be free
from dust, water droplets, and other other foreign matter; (5) before using a fresh wax, it
must be filtered in a wax oven at a temperature 2-degree Celsius higher than its melting
 point; (6) wax that has been trimmed away from the infiltrated tissue may be melted
and filtered for future use; (7) paraffin wax may be used only twice.

5. What is the effect of excessively heating the paraffin more than the recommended
temperature?

Excessively heating the paraffin more than the recommended temperature will
have the same effect as prolonged treatment in melted paraffin. Tissues become
increasingly harder and more brittle as they are heated. Therefore, overheated paraffin
will cause shrinkage and hardening of the tissues, and destroy lymphoid tissues
completely. Due to this reason, the paraffin oven should be maintained at a
recommended temperature of 2- to 5-degree Celsius above the melting point of
paraffin to be used in infiltration.

6. What is the importance of maintaining the temperature of the paraffin oven at 2-3
degrees celsius higher than the melting point of the paraffin wax?

The proper temperature of the paraffin is critical during infiltration. If the tissue is
exposed to overheated paraffin, it will cause the tissue to be brittle. However, if the
paraffin is below the recommended temperature range, infiltration of the tissue may not
be successful. Therefore, it is important that the temperature of the paraffin oven is
maintained at 2- to 3-degree Celsius higher than the melting point of the paraffin wax to
prevent relative shrinkage and hardening of the tissue. Also, maintaining the
temperature of the paraffin oven at the said range will allow proper and complete
infiltration of the tissue.

References:

Jocelyn H. Bruce-Gregorios. (2016.). Histopathologic Techniques. Katha Publishing Co.

Temperature Factor—LabCE.com, Laboratory Continuing Education. (n.d.). Retrieved
November 25, 2021, from https://www.labce.com/spg572631_temperature_factor.aspx
Tissue Clearing—LabCE.com, Laboratory Continuing Education. (n.d.). Retrieved November 25,
2021, from https://www.labce.com/spg572642_tissue_clearing.aspx