Faculty of

Medicine
Medical Education-
Damietta
University

Level 1
Semester 1
Module 1A
 ILOs
• At the end of this practical, the students should be able to:
1. Classify the different techniques for preparation of tissue
sections.
2. Enumerate types of stains and their uses.
3. Identify different types of microscopes & differences between.
4. Demonstrate and identify the cell organelles in electron
micrographs.
 Case Scenario
45 years old female patient
came with a swelling in her neck.
Her clinical examination revealed firm non-tender but fully
mobile mass. The physician asked for US imaging which
suggested that this mass may be malignant.
What will be the next step?
 What is Histology?

• The science that study the structure of the cells & tissues using
Microscope
 How do we study it?

Micro-techniques.
Micro-techniques
 Microtechniques

I-Preparing thin II- Staining of this

sections. section.
 Learning outcome 1

Classifying the different techniques for preparation of tissue

sections.
 I- Preparing thin
Sections.

1- Paraffin technique 2- Freezing method

 1- Paraffin technique.

• The routine method for histological preparation.

• Paraffin wax is used.
• Steps:
1- Obtain sample
 2- Fixation.

• Put small sample of tissue in

fixative e.g. Formalin 10%.

➢Prevents putrefaction and

autolysis.
 3-Dehydration.

• In ascending grades of alcohol

(70%-90%-100% alcohol).

➢Avoid shrinkage of the tissue.

 4-Clearing.
• With Xylol to:

➢Replace alcohol

➢Paraffin solvent

➢Renders the tissue transparent.

 5-Impregnation.
• Infiltration with Paraffin
wax in the oven.
 6-Embedding.

• In hard paraffin to
obtain a paraffin
block.
7- Sectioning .
Rotatory
Microtome
Thickness of section
is 5 um
 Paraffin technique.

1-Obtain 6-Embedding in
7-Cut section.
sample paraffin

8- Pick section
2- Fixation 5-Impregnation
on slide.

3-Dehydration 4-Clearing
 2- Freezing technique.

• Freezing of specimen by Cryocut.

❑Advantages:

1- Very rapid → rapid diagnosis in operating theatre.

2- Lipids and enzymes in the tissues are preserved →

histochemistry.
Cryocut.
 Learning outcome 2

Enumerate types of stains and their uses.

 II- Staining.

Routine histological stain→ Special stains

H&E
 Stains → dyes used for staining sections.

Acidic Basic Neutral

Leishman stain
Eosin (E) Haematoxylin (H) (Methylene blue
+Eosin)
Stain basic Stain acidic Two different
structures structures colours.
(cytoplasm) (nucleus) Acidic → basic
structures.
Acidophilic (pink) Basophilic (blue) Basic → acidic
structures.
 Staining Sections with Hematoxylin & Eosin (H&E)

Deparaffinize Mount in
in xylol Clear in Xylol Canada
balsam

Dehydrate in
Hydrate in alcohol Cover with a
alcohol (ascend.
(descend. grades) grades)
cover slip

Stain in Hx. Wash in

for 2 min distilled water

Wash with Stain in Eosin for

tap water 1 min
Hx E
Canada balsam

Cover slip
Sections stained with H&E
 Section stained with H&E

Acidophilic
cytoplasm

Basophilic
nucleus
 Special Stains.
1- Carbohydrates:

➢Periodic acid Schiff’s technique

(PAS)→ Magenta color.

➢Best's carmine (for glycogen)→

Red.
2- Lipid:

- Frozen sections are used.

✓ Sudan III → Orange.

✓ Sudan black → Black.

✓ Osmic acid → Black.

 Other methods of Stains.

Staining of living cells in vivo e.g. phagocytic cells

Vital stain by injecting Trypan blue.

Staining of living cells in vitro i.e. outside the

Supravital stain body e.g. mitochondria by Janus green B.

Metachro-matic Staining with a color different from the color of the

dye (e.g. granules of mast cells are colored red
stain when stained with toluidine blue).
 Vital stain

Macrophage with trypan

blue Tattoo
 Supravital stain Metachromatic S.

Mitochondria with Janus

green B Mast cell
 Learning outcome 3

Identify different types of microscopes.

Microscopy
The microscope is an instrument which
magnifies the image & reveals fine details of
the object.
 Microscopes Magnification up
to 400.000 times.

Light M. (Student Electron M.

M.)

Magnification 40,
100, 400, 1000, Transmission
1500. Other types
Scanning
Student Microscope
1. Eyepiece (ocular lens)
2. Revolver, or Revolving nose piece (to hold multiple
objective lenses)
3. Objective lenses
4. Coarse adjustment
5. Fine adjustment
6. Stage (to put the slide on)
7. Light source (a lamp or a mirror)
8. Diaphragm & condenser
9. Mechanical stage
 The Magnification of a Microscope

• Magnification = Power of objective lens x power of ocular lens.

Low power High power

Transmission E.M
 Transmission E.M
• Needs extremely thin sections 0.08 um.
• Electron beam is used instead of light for illumination.
• The image appears black & white.
• Dark components →
electron dense.
• White components →
electron lucent
Scanning E.M.
Give 3D images Blood
cells

Pollens
 SEM
TEM

1 2
 Other types of microscopes

➢Phase Contrast Microscope:-

• used to study the living or fixed unstained cells.

➢Fluorescence Microscope:

• used to detect fluorescent substances.

 Learning outcome 4

Demonstrate and identify the cell organelles in electron

micrographs.
 Cell Membrane

2 cell membranes. Each has trilaminar appearance

(3 layers → outer & inner electron dense layers with middle electron lucent
layer)
 Cell coat.

Moderate electron dense fuzzy coat over the surface of the microvilli of the
cells.
 Mitochondria

* Double membranous vesicle.

* The outer membrane is smooth while the inner one shows
shelf like projections called cristae.
* The cavity contains moderate electron dense matrix and
electron dense granules of calcium and magnesium.
 Rough ER

* Parallel flattened membranous cisternae with rough outer surface (due to

attached ribosomes).
 Smooth ER

Narrow anastomosing membranous tubules with smooth outer surface (free

from attached ribosomes).
 References

• Junqueira's Basic Histology Text & Atlas (15th ed.).

• Textbook of Histology, 4th ed. (Author: Leslie Gartner).
Logbook
• Check the logbook and answer the questions in this practical and sign
it from the demonstrator