Histo Tech Lab Report 2

8/12/2019 Histo Tech Lab Report 2

http://slidepdf.com/reader/full/histo-tech-lab-report-2 1/3

Hendry Rao, 012012111007, Histology Technique, Lab Report 2

LAB REPORT 2

TITLE : Dehydrating & Clearing Process

RESULTS:




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 doesn’t 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 chemicalsbecause long term effects of exposure could be devastating.




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,4th

Edition. Reprint 2010. Scion PublishingLimited,Oxfordshire, England

Bancroft J.D. 2008. Theory and Practice of Histological Techniques, Elsevier Health Sciences, 2008

Documents

Histo Tech Lab Report 2