Microtomy
Principle: For studying histological, histopathological and histochemical details of the organs and tissues thin paraffin sections are cut with a Microtome. The process is called as Microtomy.
Most
of the biopsy tests (examination of tissues) can be performed through
microtomy. By this method tissue is made fit for microscopic examination.
Microtomy
includes the following steps:
1.
Animals and tissue's for microtomy.
2.
Narcotisation of animals.
3.
Fixing of tissues.
4.
Washing of tissue.
5.
Dehydration of tissues.
6.
Clearing or dealcoholization of tissues.
7.
Embedding of tissues
.8.
Block making.
9.
Triming of blocks.
10.
Section cutting.
11.
Double staining, dehydration, clearing and mounting.
12.
Microscopic study.
Animals
for microtomy.
Frog, black rats and white or albino rats are favorite
animals for microtomy. Various tissues of these animals are used for
histological studies.
Killing
or Narcotisation.
Take a medium size frog. Chloroform it in a
large jar. Wet some Cotton in chloroforma nd put in the jar containing frog.
Close the mouth of jar with glass cover. After 15-20 minutes, when fully
anaesthetized, take out the frog and keep it in a dissecting tray. Make a
longitudinal incision in abdomen to expose viscera and collect desired tissues
of different systems or body part. .
Fixation of the
tissues.
After collection of tissues, immediately
transferred to fixative. Fixative is a chemical substance which
preserve the architecture of tissue near normal by killing cells before
enzymatic degradation by cellular
enzymes. Fixation done in a fixative for 24
hours.
Fixation
of histological samples with Bouin’s fluid
1.
Prepare 75ml saturated aqueous solution of
picric acid
2.
Add 25ml formalin (40% aqueous solution of
formaldehyde) to give 100ml total volume
3.
Add 5ml glacial acetic acid
4.
Fix tissue by submersion in Bouin’s fluid for
6 hours
5.
Transfer fixed tissue to 70% alcohol
1.
Take cleaned 100 ml wide-mouthed glass-stoppered bottles. In each
bottle keep about 50 ml . of aqueous bouin's fluid. For about 24 hrs.
(1)
Fixative renders hardness to tissues to
resist further postmortem changes.
(2)
Fixative agent coagulates and renders the
elements of tissues insoluble so that cellularsubstances may not be washed
away.
(3)
Fixative agent alters the refractive indices
of tissues and makes them optically differentiatedunder the microscope.4.
Washing
of tissues.
After 24 hours take out the pieces of tissues
and keep in a beaker. Tie the mouth of the beaker with a thin cloth and keep it
under slow running tap water. Keep on washing under tap water till all the
picric acid is removed. The indication of complete removal of picric acid comes
whenno yellowish water is seen. Normally, it takes 24 hours for perfect
washing. Tissues fixed in aqueous bouin's fluid are washed with tap water.
While those fixed in alcoholic bouin's fluid are washed with 70 percent
alcohol. While washing with 70 percent alcohol, change it frequently till the
yellow colourdisappears.5.
Dehydration
of tissues.
Dehyadration of tissue should be done through
alcoholic grades. Distilled water ( DW),
30 % , 70%, 80%, 90% and 100 % In each grade of alcohol keep
tissues for 5 to 10 minutes with 2 changes. Dehydration removes water to
prevent putrefaction. The graded alcohol slowly removes water in tissues.
Dealcoholization
or clearing.
Clearing or dealcoholizing agents is xylene used
for removal of alcohol from tissues is done through clearing agent. Take xylene
in a coupling jar and transfer tissue in
it from 100 percent alcohol. Keep tissue in xylene for 15 minutes till the
tissue become stransparent Don't leave tissue in xylene for longer period
otherwise it would become fragile. Now tissues are ready for embedding
Infiltrating:
Tissue is then infiltrating with embedding agent
like molten paraffin wax in a becker
placed in a hot air woven for 6-8
hours which replaces toluene. Others
infiltrating media are paraplust,paraplust plus, gelatin, collidins.Epoxy resin
for electron microscopy and acrylic resin for immunohistochemistry are used.
Embedding and block making . Depending on melting point of wax, adjust
the oven at 58°C or 60°C. Keep flakes of wax in a beaker of 100 ml in oven 4 to
5 hours before embedding. In another beaker keep some wax plus xylene. Now take
the tissue from xylene and first keep it in the beaker containing xylene + wax
for 30 minutes. Then transfer the tissue in pure melted wax for embedding for 45
min with two change. Normally double
time is given for embedding.
Make blocks either in metal
L-shaped angles or in paper boat or in cavity blocks. During block making, see
that no air bubble comes. If there are some air bubbles remove them with hot
spatula.
Trimming of blocks.
Trim the wax around the embedded
material and make a perfect rectangular block. On one side keep sufficient
space in the block for fixing it on a block holder. Apply half an inch wax
layer over block holder.
Sectioning : The blocks are cut by rotatory microtome. Before section cutting
the blocks are chilled in a ice because
the cold wax makes a clean cut compared to paraffin wax cut at room
temperature. The paraffin block are cut by using a rotatory microtome with 4-5 micro-mitre thickness.
Spreading : After sectioning the sectioned
tissue placed in warm water bath to floated that help to remove the wrinckles.
Making slide: Sections tissue pick up on a
glass microscopic slides having egg yolk applied for adhesion of tissue.
H E (Haemato Xylene Eosin) Technique
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