What is pathology?

It is study of disease by scientific methods.

What is disease?
Abnormal variation in structure or function induced in
body…tissue…cells by specific factors.
What is histopathology?
One of the methods used to study microscopic structural
abnormalities.
Causes of diseases.
1.Genetically determined Diseases.
2.Acquired diseases.
Chemical, physical, parasites,………………..
 Preparation of tissue for histopathological examination

Histological techniques are the methods by which cells or

tissue are prepared to demonstrate morphology by dye
staining. The techniques used for preparation of
materials for microscopic examination include sectional
and non-sectional methods. Sectional method involves
cutting the specimen into very thin parts or sections
using an instrument known as a microtome. Specimen is
then processed (fixed, dehydrated, stained) and
examined under light microscope
1.Fixation
Fixation is a complex series of chemical events which prevents postmortem degeneration
and other misleading structural changes in cells and tissues. It preserve cells and tissue
constituents in a condition identical to that existing during life.
Aim of Fixation:-
1.Fixation prevents autolysis which is caused after the death of cells by the action of intercellular
enzymes released when cells die.

2.Fixation also prevents bacterial decomposition which causes changes in the tissues that are
very similar to those of autolysis and is brought about by the presence of multiplying bacteria
in the diseased tissues at time of death.

3.Fixatives coagulate proteins which are present in great abundance in cells and tissues.

4.Fixation prevents loss of easily diffusible substances.

5.Fixation is important to fortify the tissue against the deleterious effects of the various stages in
the preparation of sections.

6.Fixation leaves the tissues in a condition which facilitates differential staining with dyes and
other agents.
Examples of fixative solutions
Bouin’s Fluid :-
Saturated aqueous picric acid 75 ml
Formalin 25 ml
Acetic acid 5 ml
Blocks are fixed for 6-24 hours and transferred to 70 per cent alcohol. The yellow
staining of tissues should be removed from sections by treatment with alcohol
followed by 2.5 per cent sodium thiosulphate.
Neutral buffered formaldehyde :
Sodium phosphate, mono basic 4g
Sodium phosphate , dibasic 6.5g
Dissolve in water to 1000 ml
Heat to boiling ,
add 25-40g of a paraformaldehyde powder ( in fume hood),
stir well, cool, and filter. Keeps for several weeks.
10% formalin :
Formalin 100 ml
Sod.chloride 8.5 g
Water 900 ml
• 2. Dehydration
• In the fixation and post fixation treatment ,
water is used even in fixatives or as running tap water in
post fixation . But paraffin wax will not penetrate tissues
in the presence of water , so there must be a process in
which the water removed from the tissue completely this
process called “ dehydration “ , this is effected by
immersion of the tissue in ethyl alcohol and it is usual to
begin with a dilution of alcohol in water e.g. 70 per cent
alcohol. A commonly used range of dehydrating
solutions is 70 per cent, 90 per cent, and two or three
changes of absolute alcohol .
• 3. Clearing
• The alcohol does not mix with wax, and
for this it must be removed to allow wax
infiltration. This can be done by placing
the tissue in a chemical that is miscible
with molten wax. There are a number of
clearing agents available such as xylene,
toluene, benzene, chloroform and cedar
wood oil.
• 4. Embedding
Having been completely
impregnated with wax , it is necessary to obtain a solid
block containing the tissue . This is done by filling a
mould of suitable size with molten paraffin wax ,
orientating the specimen in the mould to ensure its being
cut in the right plane , and finally cooling the mass to
promote solidification.
Embedding containers :
A- Glass dishes
B- Metal L-shape
C- Paper “boats”
5. Sectioning
After embedding, sections are prepared on an instrument known as
a microtome. The thickness of sections produced during microtomy may rang
between 3-8 microns. There are 5 types of microtomes available :

1.Cambridge rocking microtome

2 - Rotary microtome

3-Sliding and base sledge microtome

4-Freezing microtome

5-Ultramicrotomes
Affixing the Sections
Before further processing, the paraffin sections are attached to
glass microscope slides. This process is referred to as
affixation.
Two of the most widely used affixatives are Haupt's and Mayer's.
Haupt's Affixative
Distilled water 100 ml
Gelatin 1 gm.
Phenol crystals 2 gm.
Glycerin 15 ml.
Mayer's Affixative
Egg albumen 5 ml.
Glycerine 50 ml.
Sodium salicylate 1 gm.
6. Staining
. Two stains are used for histopathological technique , haematoxylin and eosin stains.
Haematoxylin stains:
Haematoxylin is the most widely used and versatile dye in histological technique. It is a basic natural dye
extracted from the wood of the tree Haematoxylin campechianum and is used in stains for the demonstration
of cell nuclei, myelin, elastic fibers, fibrin, neuroglia, and muscle striations.
1.Ehrlich’s haematoxylin
Solution A
Haematoxylin 2g Dissolve
Absolute alcohol 100 ml
Solution B
Glycerine 100 ml
Distilled water 100 ml
Aluminium potassium sulphate (10-14 g)
(potassium alum)
Mix A and B together and add 10 ml glacial acetic acid. Ripens in approximately 8 weeks, when it turns dark red.
2.Harris’ haematoxylin
Preparation:
Haematoxylin 1g
Absolute alcohol 10 ml
Ammonium or potassium alum 20 g
Distilled water 200 ml
Mercuric oxide 0.5 g
Eosin stain:
Eosin, a synthetic acidic red dye, properly used on well fixed
material, stains cytoplasm in varying intensity and shades of the
primary colour giving a most useful differential stain.
a.1% Aqueous Eosin Solution
Eosin Y, water soluble 10 g
Distilled water 1000 ml
Dissolve and add:
Glacial acid 2 ml
b. 1% Alcoholic Eosin Solution
Eosin Y, water soluble 1g
Distilled water 20 ml
Dissolve and add:
Alcohol, 95% 80 ml
 GENERAL METHODS OF STAINING
Haematoxylin and eosin technique
1- Place slide in xylene to dewax 5-10 min
2- Absolute ethanol 90‫و‬% ethanol 70 ‫و‬% ethanol 1-2 min
3- Ehrlich’s haematoxylin (to stain nuclei only) 10-15min
4-Blue in alkaline tape water (weak bicarbonate solution) for 10-15
min
5- Examine ( x 5 or x10 objective of microscope, wipe underside of
slide and do not allow it to dry up) .Remove excess stain in acid
alcohol ( 2-3 min) .
6- 70% ethanol 2 min
7- Counterstain in 0.05% alcoholic eosin 3-5 min.
8- Dip in 70% ,80%,90% and absolute ethanol 2-3 min
9- Clear in xylene ( not the one used for dewaxing) 5 min
10- Mount in one drop of D.P.X.