Pathology PSW-16

1- Histology
and
Histo-technique
MICROSCOPY
- Light microscope
- Electron microscope: Scanning / Transmission
- Fluorescence microscope
- Inverted microscope
- Phase contrast microscope

Light microscope
Light microscopic pictures
Electron microscopic picture
Tissue Preparation for Light Microscope
Tissue specimens received in the surgical pathology
laboratory have a request form that lists the
patient information and history along with a
description of the site of origin.

1- Fixation
• The purpose of fixation is to preserve tissues permanently in
as life-like state as possible.
• Fixation should be carried out as soon as possible after
removal of the tissues to prevent autolysis.
• There is no perfect fixative, though formaldehyde comes the
closest.
• Therefore, a variety of fixatives are available for use,
depending on the type of tissue present and features to be
demonstrated.
Types of fixatives
• There are five major groups of fixatives,
classified according to mechanism of action:
• Aldehydes
• Mercurials
• Alcohols
• Oxidizing agents
• Picrates
• Aldehydes:
Include formaldehyde (formalin) and glutaraldehyde.
• It is good for immuno-histochemistry techniques.
• Formalin penetrates tissue well, but is relatively slow.
• The standard solution is 10% neutral buffered
formalin.
• Mercurials fix tissue by an unknown mechanism.
• They contain mercuric chloride and include such
well-known fixatives as Zenker's.
• These fixatives penetrate relatively poorly and
cause some tissue hardness, but are fast and give
excellent nuclear detail.

• Alcohols: including methyl alcohol (methanol)
and ethyl alcohol (ethanol).
• However, they are very good for cytologic
smears because they act quickly and give good
nuclear detail.
• Oxidizing Agents:
Include permanganate fixatives (potassium
permanganate), dichromate fixatives (potassium
dichromate), and osmium tetroxide.
• Picrates: include fixatives with picric acid.
• Foremost among these is Bouin's solution.

2-Factors affecting fixation
There are a number of factors that will affect the fixation
process:
• Buffering: Fixation is best carried out close to neutral
pH, in the range of 6-8.
• Penetration of tissues depends upon the diffusability of
each individual fixative, which is a constant.

• The volume of fixative is important. There should be a
10:1 ratio of fixative to tissue.
• Increasing the temperature, as with all chemical reactions,
will increase the speed of fixation.
• Concentration of fixative should be adjusted down to the
lowest level possible.
• Time interval: Also very important is time interval

from removal of the tissues to the fixation.
Tissue Processing
• The technique of getting fixed tissue into
paraffin is called tissue processing. The
main steps in this process are
dehydration and clearing.

1. Dehydration: Gradual removal of water from
the tissue using ascending grads of ethyl alcohol
to prevent tissue shrinking.
2.Clearing: Replacement of alcohol in tissue by
clearing fluid like xylene, benzene, or acetone.

3. Embedding:
- Tissues are impregnated in paraffin
4. Cutting:
- Paraffin block are cut by microtome using metal

knife, into thin sections ~ 6µ
6. Mounting:
- Sections spread on the hot plate and mounted on
glass slides.
7. Staining:
- Variable stains are used for specific tissues.
Automated tissue
processor
Sectioning
• Once the tissues have been embedded, they must
be cut into very thin sections (4 to 6 microns)
that can be placed on a slide.
• This is done with a microtome. The important
thing for proper sectioning is a very sharp knife.

• Frozen sections are performed
with an instrument called a
cryostat.
Frozen Sections • The cryostat is just a
refrigerated box containing a
microtome.
• The temperature inside the
cryostat is about -20 to -30 C.
• The tissue sections are cut and
picked up on a glass slide.
• The sections are dried and then
stained.
Staining
The embedding process must be reversed in order to get
the paraffin wax out of the tissue and allow water soluble
dyes to penetrate the sections.
Therefore, before any staining can be done, the slides are
"deparaffinized" by running them through xylene then,
to alcohols and lastly to water.
There are no stains that can be done on tissues containing
paraffin.
Automated stainer
Frozen sections are stained

by hand, because this is
faster for one or a few
individual sections.
Coverslipping
The stained section on the
slide must be covered
with a thin piece glass to
protect the tissue from
being scratched, and to
preserve the tissue section
for years to come.
Decalcification
• Bone specimens as well as calcified tissues are the
most type here.
• The calcium must be removed before embedding
to allow sectioning.
• A variety of reagents have been used to decalcify
tissue such as mineral acids, organic acids, EDTA,
and electrolysis.
 Strong mineral acids such as nitric and
hydrochloric acids
 Strong acids will remove large quantities of
calcium at a rapid rate, but they will cause
damage of cellular morphology.

• Organic acids such as acetic and formic acid.
• However, they act more slowly on dense cortical
bone.
• EDTA can remove calcium safely, it works slowly, it
penetrates tissue poorly, but it is expensive in large
amounts.

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1- Histology

- Electron microscope: Scanning / Transmission

- Phase contrast microscope

Tissue specimens received in the surgical pathology

laboratory have a request form that lists the

patient information and history along with a

description of the site of origin.

Include formaldehyde (formalin) and glutaraldehyde.

• It is good for immuno-histochemistry techniques.

• Formalin penetrates tissue well, but is relatively slow.

• The standard solution is 10% neutral buffered

• They contain mercuric chloride and include such

well-known fixatives as Zenker's.

• These fixatives penetrate relatively poorly and

cause some tissue hardness, but are fast and give

excellent nuclear detail.

and ethyl alcohol (ethanol).

• However, they are very good for cytologic

smears because they act quickly and give good

Include permanganate fixatives (potassium

permanganate), dichromate fixatives (potassium

dichromate), and osmium tetroxide.

• Picrates: include fixatives with picric acid.

• Foremost among these is Bouin's solution.

• Buffering: Fixation is best carried out close to neutral

pH, in the range of 6-8.

• Penetration of tissues depends upon the diffusability of

each individual fixative, which is a constant.

10:1 ratio of fixative to tissue.

• Increasing the temperature, as with all chemical reactions,

will increase the speed of fixation.

• Concentration of fixative should be adjusted down to the

lowest level possible.

• Time interval: Also very important is time interval

• The technique of getting fixed tissue into

paraffin is called tissue processing. The

main steps in this process are

dehydration and clearing.

the tissue using ascending grads of ethyl alcohol

to prevent tissue shrinking.

2.Clearing: Replacement of alcohol in tissue by

clearing fluid like xylene, benzene, or acetone.

- Paraffin block are cut by microtome using metal

be cut into very thin sections (4 to 6 microns)

that can be placed on a slide.

• This is done with a microtome. The important

thing for proper sectioning is a very sharp knife.

Frozen sections are stained

 Strong acids will remove large quantities of

calcium at a rapid rate, but they will cause

damage of cellular morphology.

• However, they act more slowly on dense cortical

• EDTA can remove calcium safely, it works slowly, it

penetrates tissue poorly, but it is expensive in large

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