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1. MUNI UNIVERSITY
FACULTY OF SCIENCE
EDUCATION DEPARTMENT
BIO2203
• Group members
• Nakusi Gloria 2001201041
• Adomati Oresto 1901200057
• Baramas Ronny Towet 2001201016
• Nyesika Abel

2. OBTAINING SAMPLE FOR
HISTOLOGY
Tissues Collection
• The tissue is removed from the animal body or plant
part.
• Tissue submitted for histopathology must not be
more than 1cm3 thick (ideal size).
• Most specimens from solid tissues are cut in the
form of pieces measuring 10 to 15 mm on the slides
and 2 to 3 mm in thickness.
• Small fragments of tissue must be wrapped in thin
paper.
• Placed in a fixative which stabilizes the tissues to
prevent decay The most common fixative is
formalin (10% formaldehyde in water)

3. Illustration

4. • This will slowly penetrate the tissue causing
chemical and physical changes that will
harden and preserve the tissue and protect it
against subsequent processing.

5. • Fixation should be carried out as soon as
possible after removal of the tissues or soon
after death to prevent autolysis. There is no
perfect fixative.
• The fixatives depend on the type of tissue
present.

6. Importance Of fixation
• To prevent autolysis and bacterial attack. To
fix the tissues so they will not change their
volume and shape during processing
• To prepare tissue and leave it in a condition
which allow clear staining of sections
• To leave tissue as close as their living state
as possible, and no small molecules should
be lost.

7. • Processing of Tissue
Embed the tissue in a solid medium firm
enough to support the tissue and give it
sufficient rigidity to enable thin sections to
be cut and yet soft enough not to
damage the knife or tissue.
Stages of processing
 Dehydration, Clearing, Infiltration &
Impregnation, Embedding, Sectioning,
Staining, Mounting

8. Dehydration
• Water is completely removed from fixed tissue
Procedure
• Tissue blocks are place in capsules
(Cassettes)
• These tissues are passed through a series
of increasing concentration of alcohol
• 2 bath in 80 % alcohol for 1hour
• 1 bath in 90 % alcohol for 1hour
• 3 bath in 100 % alcohol for 1hour

9. Clearing
An intermediate solvent that is fully miscible with
both ethanol and paraffin wax is used. This solvent
will displace the ethanol in the tissue, then this, in
turn, will be displaced by molten paraffin wax. This
stage in the process is called “clearing” and the
reagent used is called a “clearing agent”. Reagents
are; Xylene, Chloroform, Benzene, Carbon
tetrachloride
Procedure
• 2 bath in Clearing agent for 1hour

10. Infiltration And Impregnation
• Clearing agent is eliminated from the tissue by
diffusion into the surrounding melted wax
(infiltration)
• A typical wax is liquid at 60°C and can be
infiltrated into tissue at this temperature then
allowed to cool to 20°C, where it solidifies to
a consistency that allows sections to be
consistently cut.

11. Embedding Or Blocking Out
• The infiltrated and impregnated tissue is placed
in warm quid paraffin, which forms a firm
block after cooling.
Procedure
• Paraffin wax is first melt 56°C to 58°C and
filter through course filter paper. Fill mold
with paraffin. Place tissue in mold
• The mold is then placed in a container of cold
water or kept in refrigerator for 10 to 20
minutes.

13. • Sectioning
• Cut biological specimens into very thin
sections for microscopic
• Procedure
• Trim the block with a knife until 1 to 3mm of
paraffin remains all side of the tissue.
• Fix the block in the block holder of the
microtome. Insert tightly a knife in the knife
holder with proper position.
• Cut sufficient sections.

14. Frozen Section
• The frozen section is a technique in which
tissue is frozen rapidly to the temperature of
-20°C and the sections are cut and stained.
• Tissue can be examined microscopically
within 5-10 minutes of its removal from the
body
• It reduces the time of processing from 18
hours to 5 minutes.

15. Staining
Staining is used to highlight important features
of the tissue as well as to enhance the tissue
contrast.
The most common stain applied for histological
study is Haematoxylin and Eosin.
Washing and rinsing of tissue sections is a
necessary part of most staining techniques.
It eliminates carrying over of one dye solution to
the next.

16. Cover slipping and mounting
Hold the slide between the thumb and the forefinger of
one hand and wipe with a clean cloth both ends of the
slides. Look for the engraved number to make sure the
side the sections is present.
Clean carefully around the section and lay on a clean
blotting paper with section uppermost along with
appropriate coverslip which has already been polished.
Place a drop of mountant on the slide over coverslip.
Amount of mountant should be just enough. Invert the
slide over the coverslip and lower it so that it just
adheres to the cover slip quickly turn the slide over,
then lay it on a flat surface to allow the mountant to
spread. Do not press or push the slide at all. It can
damage the section.