Hendry Rao, 012012111007, Histology Technique, Lab Report 2

LAB REPORT 2
TITLE : Dehydrating & Clearing Process
RESULTS:







Hendry Rao, 012012111007, Histology Technique, Lab Report 2

DISCUSSION:
Fixatives, such as formalin, will react with any staining procedure. So after the fixation process, the
tissue is normally flushed to remove the fixative residue. To ensure the infiltration process of the paraffin wax,
all liquids have to be removed in this case water. This is achieved in dehydration process.
A serial dilution of increasing alcohol concentration was used, starting from 70% all the way to 100%. A
tissue should not be allowed to air dry. This is to avoid it from distorting the cell morphology. The aim of
dehydration is to remove all traces of water from the tissue without causing maximum damage to the tissue
and its cells. Air drying would do the opposite. It would cause the tissue to dry up and the tissue to shrink n and
distort. This is a chemical process and never a physical process.
Dehydrating agents that are very commonly used are ethanol and acetone. The obvious
disadvantage of this procedure is the shrinkage of the tissues used, removal of organelles in the cell
and as well as causing the cells to rupture or crenate if improper technique is done. So, dehydration
is done in succession of serial dilution of increasing concentration.
Clearing is the transition step before infiltration. Many dehydrants are nor soluble with paraffin
wax, and a clearing agent must be used to enable miscibility to occur. Shrinkage may occur so this
stage reduces the likelihood of this. At this stage, the tissue would shrink for the final time as fats from
the tissue would be fully dissolved. Clearing also allows the tissue to be translucent. Even it is
translucent, this doesnt affects the tissue ability to be chemically active towards routine H & E stain
as well as special stains.
Clearing agents are chosen primarily, xylene because of its reactivity towards the type of
tissues to be processed, and the type of processing to be undertaken. The temperature used is also
quite stable and would perform well in vacuum and as well as pressure. Besides that, it is also very
cost effective. A few safety procedures should be taken into account. As these agents of dehydrating
and clearing would easily form fumes, so the procedure should be carried out in a fume hood or
properly ventilated lab. Also gloves should be worn at all times when handling these chemicals
because long term effects of exposure could be devastating.



Hendry Rao, 012012111007, Histology Technique, Lab Report 2

CONCLUSION:
The technique of dehydration and clearing process as well as identification of types of media and its
used was learned.

QUESTION:
1. What is the purpose of dehydration process ad state the dehydrants media used in this
process?
The dehydration process is done to remove water from the tissue to enable infiltration of
paraffin wax. The media used are Serial Diluted alcohol of increasing concentration from 70%,
80%, 90% and absolute alcohol.

2. What is the purpose of clearing process and state the clearing agent or media used in
this process?
Clearing process is done to remove the alcohol from dehydration process and enable the
paraffin wax to infiltrate the tissue to give it structural support.

3. List out the characteristics of choosing clearing agent.
Rapid removal of dehydrating agents
Minimal tissue damage
Less toxic
Flammability Index
Cost effective
REFERNCES
Kernian J.A,2008.Histological and Histochemical Methods,4
th
Edition. Reprint 2010. Scion Publishing
Limited,Oxfordshire, England
Bancroft J.D. 2008. Theory and Practice of Histological Techniques, Elsevier Health Sciences, 2008