Histology
Department
FHB Module
Practical I
OF MEDICINE
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Microtechniques &
Microscopes
Histology
Department
Intended learning outcomes (ILOS)
• Comprehend the basic steps of tissue preparation for
microscopic examination using paraffin technique.
• Identify instruments and chemicals used in preparation and
sectioning of paraffin blocks and staining of tissues .
• Compare the different types of commonly used microscopes.
• Discriminate between photomicrographs of light microscope,
fluorescence microscope, SEM and TEM.
MICROTECHNIQUES
Histology
Department
• Paraffin technique: most rapid (routine).
• Celloidin technique: most ideal (obsolete).
• Freezing technique: suitable for protein
detection (Surgical pathology & research.
Histology
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Paraffin Technique
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✓Samples
may be obtained in surgical
operations, biopsy, autopsy or blood sample
(film).
✓Small pieces of about 3mm of tissue are cut
from the specimen and placed into cassettes,
then rinsed in a fixative.
Histology
Department
Histology
Department
Specimens are usually fixed in 10% buffered formalin
for several hours to:
➢denature the proteins and harden them.
➢prevent
further decomposition of the
arresting cell lysis.
tissue by
Histology
Department
Histology
Department
Tissues must be dehydrated before they can
be infused with paraffin.
✓
✓ Tissue is to be submerged in ascending series
of ethyl alcohol baths to remove water
gradually.
Histology
Department
✓The alcohol must then be cleared with a clearing
agent, such as xylene or tolune, which are
miscible with paraffin.
✓This process is performed overnight so that the
tissue is ready to be embedded with paraffin the
next morning.
Histology
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✓The tissue is placed in molten
paraffin at 52- 56°C for several
minutes up to overnight
incubation.
✓Paraffin
is left at room
temperature to cool so that the
tissue and block will be hard
enough to be cut.
‫مشاهدة الصورة بالحجم الكامل‬
Histology
Department
Histology
Department
➢ The cut sections are floated on a warm water bath to
remove wrinkles, then transferred by hand or soft brush
to glass slides.
paraffin blocks containing the tissue are cut
producing 5µm sections using a microtome. This piece
of equipment has a set mechanism for advancing the
block across a very sharp knife.
➢ The
File:Tissue processing - Solid paraffin block is mounted on the tissue holder of a microtome.jpg
Histology
Department
Histology
Department
Histology
Department
Histology
Department
For the water-soluble dyes to penetrate the tissue,
the sections must be rehydrated.
Xylene is used to dissolve the paraffin, then a
series of descending grades of ethyl alcohol
washings rehydrates the tissue.
Histology
Department
➢ The
slides are stained to differentiate the
nuclear material and connective tissue from the
rest of the tissue components.
➢ The
regularly-used hematoxylin-eosin (H & E)
stain is used by most laboratories.
Histology
Department
Histology
Department
✓The tissue is again dehydrated with alcohol and cleared
with xylene then mounted in canada balsam to preserve
it indefinitely.
✓A
glass cover slip is applied manually or by an
automated machine.
✓The
slides are now ready to be viewed by the
pathologist.
Histology
Department
Recommended Videos
• https://www.youtube.com/watch?v=KnMdSgd5mts
• https://www.youtube.com/watch?v=3PBoHU_pidQ&t=155s
Histology
Department
MICROSCOPES
• Microscopes are instruments designed to produce
magnified visual or photographic images of small
objects.
The microscope must accomplish three tasks
1. produce a magnified image of the specimen
2. separate the details in the image,
3. render the details visible to the human eye or
camera.
Compound Light Microscope
• Lets light pass through
an object and then
through two or more
lenses.
Histology
Department
Compound Light Microscope
• Lets light pass through
an object and then
through two or more
lenses.
Histology
Department
Histology
Department
FLUORESCENCE MICROSCOPE
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INVERTED (Phase Contrast) MICROSCPE
Electron microscopes
Use a beam of electrons instead of a beam
of light to magnify the image.
Histology
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Histology
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Transmission electron
microscopy (TEM)
• Allows the observation of
molecules within cells
• Allows the magnification
of objects in the order of
100, 000’s.
Histology
Department
Transmission electron microscope (TEM)
– Provides for detailed study of
the internal ultrastructure of
cells
– a beam of electrons
is transmitted through
the specimen for
a 2D view
Histology
Department
Scanning electron microscope (SEM)
– Study of the external
appearance of cells
– a beam of electrons
scans the surface of
the specimen for
a 3D view
Histology
Department
IMPORTANT CONCEPTS
Magnification Power
Degree of enlargement.
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Resolving Power
The least distance between
two points to be seen as two
and not one (Sharpness).
• Occular X Objective
• Unite= diopeter (X)
• NE= 0.2 mm
• LM= 0.2 µm
• EM= 0.2 nm
Histology
Department
Histology
Department
Histology
Department
SELF ASSESSMENT
Histology
Department
1) What is the importance of using fixatives ?
2) What is the advantage of using parrafine technique ?
3) Explain why alcohol is used in ascending concentration?
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4) What is the type of microscope used to obtain this photo?
A) Scanning electron microscope
B) Light microscope
C) Transmission electron microscope
D) Fluorescence microscope
Histology
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5) What is the type of microscope used to obtain this photo?
A) Scanning electron microscope
B) Light microscope
C) Transmission electron microscope
D) Fluorescence microscope
Histology
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5) What is the type of microscope used to obtain this photo?
A) Scanning electron microscope
B) Light microscope
C) Transmission electron microscope
D) Fluorescence microscope
Histology
Department
5) What is the type of microscope used to obtain this photo?
A) Scanning electron microscope
B) Light microscope
C) Transmission electron microscope
D) Fluorescence microscope
Histology
Department
A first year medical student was
examining a histological slide of a
section in skeletal muscle. He used a
10X eye piece and a 40X objective
lens. What is the magnification of the
observed image?
Answer: 400 times
Histology
Department
THANK YOU