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MODULE
Tissue Processing
Histology and Cytology
7
TISSUE PROCESSING
Notes
7
...
This describes the steps required to take animal and human tissues from fixation
to the state where it is completely infiltrated with a suitable wax i
...
paraffin wax
and can be embedded and ready for section cutting on microtome
...
Aim: To process the fixed tissue into a form in which it can be made into thin
microscopic sections
...
Dehydration: It is the process of removing water from tissues
...
Dehydration is usually complete
when less then 3-4% of water remains in the tissues
...
Permeability of tissues
2
...
Temperature
4
...
The most commonly used dehydrant is ethyl alcohol
...
Concentration of first alcohol bath depends on the
fixative and size and type of the tissue, e
...
delicate tissue needs lower
concentration of alcohol and smaller interval between two strengths of alcohol
...
After about 40 tissues have passed through the first change of alcohol,
it is discarded and all the other changes are brought one step lower
...
Usually tissues are kept in each solution for 40 to 60 minutes
...
Anhydrous CuSO4
removes water from alcohol as it in turn removes it from tissues
...
Therefore,
it acts as an indicator for the presence of water
...
Rapid dehydration
2
...
Blue colouration of CuSO4 indicates that both alcohol and CuSO4 should
be changed
...
It is used for complete dehydration
...
Advantages
z
Rapid action
z
Easily removed by most clearing agents
z
Less expensive
Disadvantages
z
Highly volatile
z
Causes shrinkage and brittleness of tissues
z
Dissolves lipid more than ethanol
Clearing – Clearing is a process which leaves the tissues clear and transparent
...
If the refractive index of the clearing agent is similar to the protein
of tissue the tissue becomes transparent
...
Thus clearing serves two purposes
MODULE
Histology and Cytology
1
...
Acts as solvent for the mounting media which renders the tissues transparent
and improves the refractive index, making microscopic examination easier
...
Two changes of one hour
each are given to get the end point
...
It is not preferred for brain tissue
...
7
...
During this process clearing agent diffuses out and molten wax
is infiltrated
...
This
process is called impregnation
...
The duration and number of changes required for
thorough impregnation of tissue depends on 1
...
Vacuum
reduces the time required for complete impregnation
...
Clearing agent employed
3
...
Routinely 12 containers containing different solutions are used
for processing in the following order
HISTOLOGY AND CYTOLOGY
37
MODULE
Histology and Cytology
Notes
Tissue Processing
z
10% formalin – container no 1, 2
z
50% alcohol – container no 3
z
90% alcohol – container no 4 & 5
z
Absolute Alcohol – container no 6
z
Acetone – container no 7 & 8
z
Xylene – container no 9 & 10
z
Paraffin Wax – container no 11 & 12
INTEXT QUESTIONS 7
...
In tissue processing the tissues are impregnated into
...
Steps in tissue processing are
...
Process of removing water from tissue is
...
Most commonly used dehydrant in tissue processing is
...
Usually
...
...
Clearing is the process which leaves the tissues
...
Clearing serves two purposes namely
...
Most commonly used clearing agent is
...
The process by which the infiltrated tissue gets deposited is called
...
3 TISSUE PROCESSING (PRACTICAL-1)
Tissue processing can be performed either manually or through automated tissue
processor
...
Two types of devices are available
z
Tissue transfer or dip dunk
z
Fluid transfer or enclosed
Advantages of automated tissue processor - Saves time, decreases human error,
effective fluid circulation, Temperature can be adjusted and vacuum/pressure
can also be incorporated
...
Small blocks of tissue are enclosed in the perforated capsules
...
When the superstructure
descends the basket is immersed in the first solution and other reagent beakers
are covered preventing evaporation of reagents
...
During immersion the basket rotates so
the infiltration of fluid into the tissues is optimum
...
The machine is started in the evening so that the process is complete
in the morning,and embedding is done
...
7
...
Manual
In this process the tissue is changed from one container of reagent to another
by hand
...
7
...
Labels should be written with graphite pencil, India ink or typed
...
The fluid used in complete dehydration and clearing tend to become
contaminated with fluid carried over from previous vat by the tissue
...
Other reagents are
changed twice a week or earlier with an average work load
...
WHAT HAVE YOU LEARNT
z
40
The technique of getting tissues fixed into paraffin is called tissue
processing so that thin microscopic sections can be achieved
...
z
Most commonly used dehydrant is ethyl alcohol
z
Anhydrous copper sulphate removes water from alcohol as it inturn removes
it from tissues and acts as a indicator for the presence of water
z
Acetone is used for complete dehydration
z
Clearing is the process which leaves the tissues clear and transparent
z
Clearing serves two purpose as it removes alcohol to make paraffin
impregnation complete and acts as a solvent for mounting media
z
Xylene is the most commonly used clearing agent
z
Impregnation is the process by which the infiltrated wax gets deposited
MODULE
Histology and Cytology
Notes
TERMINAL QUESTIONS
1
...
Explain clearing process of tissue processing
3
...
1
1
...
Dehydration and clearing
3
...
Ethyl alcohol
5
...
Anhydrous copper sulphate
7
...
Removes alcohol and acts as a solvent
9
...
Impregnation
HISTOLOGY AND CYTOLOGY
41