Preparation of Tissue for Microscopic

Viewing – Histological Staining

Histology

With Professor Geoffrey Meyer

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Learning Objectives

At the end of this lecture you should…

• …appreciate the techniques involved in

preparing a piece of tissue/organ for light
microscopic viewing.

• …understand the chemical basis of

histological staining.

• …know what the terms “basophilia” and

“acidophilia” mean.

• …be familiar with H&E staining and some

other common histological stains and what
cell/tissue components they help to
visualize.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains

• Preparation of tissue for microscopic

examination

• Common histological stains and

reactions

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Processing of Tissues

In brief, the "routine" sequence of events is as

follows:

1. Obtain a piece of normal human or animal tissue.

2. Fix it for 24 hours or more in an appropriate

fixative.

3. Dehydrate it through an increasingly higher

concentration of alcohols.

4. Replace alcohol ("clear") with xylol or chloroform.

5. Infiltrate it with paraffin (or celloidin).

6. Embed it in a block of paraffin (or celloidin).

U.S. Fish and Kevin

WildlifeBocanegra
Service Northeast
Alvarado,Region, PD;
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Processing of Tissues

In brief, the "routine" sequence of events is as

follows:

7. Cut the tissue in thin sections on the microtome

(5 to 10 μm thick).

8. Mount the sections on glass slides.

9. Remove (dissolve) the embedding medium.

10. Rehydrate the sections in decreasing

concentrations of alcohols.
11. Stain the sections with an appropriate staining
sequence.

12. Again dehydrate the sections in increasing

concentrations of alcohols.
U.S. Fish and Kevin
WildlifeBocanegra
Service Northeast
Alvarado,Region, PD;
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Processing of Tissues

In brief, the "routine" sequence of events is as

follows:

13. Clear the sections in xylol.

14. Attach the protective coverslip with a mounting

medium.

15. Label the slide.

16. View the section.

U.S. Fish and Kevin

WildlifeBocanegra
Service Northeast
Alvarado,Region, PD;
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Hematoxylin and Eosin

Most commonly used dyes in histology:

Hematoxylin:
Is a basic dye and has a net positive
charge on its colored portion [Dye+Cl-]

Eosin:
Is an acidic dye and has a net negative
charge on its colored portion [Na+Dye-]

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains (Basophilia)

Basic dyes react with anionic components of cells and tissues

(i.e. those carrying a net negative charge).

Basophilia – The reaction varies with pH.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains (Basophilia)

Some anionic components are:

Carboxyl groups of proteins pH = about 10

Phosphate groups of nucleic acids pH = 5–7 i.e. slightly acidic to neutral

Sulphate groups of glycosaminoglycans pH = 4 or less

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains (Acidophilia)

Acid dyes react with cationic components of cells and tissues,

especially the ionized amino groups of proteins.

Acidophilia – Reactions are not as specific as reactions with

basic dyes.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains (Hematoxylin and Eosin = H&E)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Basophilia

A limited number of substances within cells

and the extracellular matrix display basophilia:

• Heterochromatin and nucleoli (ionized

phosphate groups in nucleic acids)

• Ergastoplasm (ER) and ribosomes (ionized

phosphate groups in ribosomal RNA)

• Extracellular material e.g. matrix of

cartilage (ionized sulphate groups in
nucleic acids)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Acidophilia

Staining with acid dyes is less specific but

more substances within cells exhibit
acidophilia:

• Cytoplasmic filaments (e.g. contractile

filaments in muscle cells)

• Intercellular membranous components

and unspecialized cytoplasm

• Extracellular fibers (ionized amino acid

groups)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Cytoplasmic Filaments in Skeletal Muscle Fibers

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Histological Stains (Hematoxylin and Eosin = H&E)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 H&E Staining Has Limitations

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Differential Staining – A Trichrome Stain

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Masson’s Trichrome

This is often used to stain connective tissue. Trichrome

means the technique produces three colors. Nuclei and
other basophilic (basic-liking) structures are stained blue,
cytoplasm, muscles, erythrocytes and keratin are stained
bright red. Collagen is stained green or blue, depending on
which variant of the technique is used.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Masson’s Trichrome

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Differential Staining – A Trichrome Stain

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Silver Stain

• Sometimes it is used to
demonstrate fine structures such
as cell processes in neurons.

• It produces a black, brown or

golden stain.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Bielschowsky (Silver Stain)

• It can be used to visualize

nerve fibers.

• The Bielschowsky technique is

a silver staining method used
for the visualization of nerve
fibers, including multipolar
interneurons in the cerebellum.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Myelin

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Osmium Stain

These dyes stain lipid-

containing structures such as
myelin in a brownish black
color.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Osmium Stain

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Kluver (Luxol Fast Blue) Stain Commonly Counterstained with H&E

• Myelin fibers: blue to blue/green

• Neurons: violet
(when counterstained)

• Red blood cells: blue

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Van Gieson Stain

F.l.t.r.: © by Geoffrey Meyer, Meyer’s

Kevin Bocanegra Histology,
Alvarado, (http://histology-online.com); Internet Archive Book Images, PD
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Reticulin Stain

Stains reticular fibers blue/black

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Elastic Stain

Stains elastic fibers blue/black

in connective tissues (and the
wall of the aorta)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Periodic Acid-Schiff (PAS) Reaction

Periodic acid-Schiff (PAS) reaction stains

carbohydrates and carbohydrate rich
macromolecules, e.g. glycogen in cells,
mucus in cells, basement membranes
underlying cells and reticular fibers in
connective tissue.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Giemsa Stain

• Giemsa is usually used for staining

blood and bone marrow smears.

• Nuclei are stained dark blue to

violet, cytoplasm pale blue,
erythrocytes pale pink.

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 What Is “Resolving Power”?

Resolving power is the ability of a microscope lens (or optical

system) to clearly distinguish 2 closely positioned objects.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Resolving Power

“Resolution” depends on the optical system, the wavelength of light (or electron beam) and:

r = minimum distance between λ = the wavelength of light or emission

2 resolvable objects wavelength (electron beam) etc.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Distance between Resolvable Points

Human eye 0.2 mm or 200 micrometers

Light (bright field)

0.2 µm (0.2 micrometers)
microscope

Transmission electron
1.0 nm (1 nanometer)
microscope

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Electron Microscope

Better resolution provides cellular structural details

Brush border = Microvilli

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Electron Microscope

Better resolution provides cellular structural details

Brush border = Microvilli

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 Electron Microscope

Better resolution provides cellular structural details

Sarcomere (skeletal muscle contractile unit)

© by GeoffreyKevin
Meyer, Meyer’s Histology,
Bocanegra Alvarado,(http://histology-online.com)
kevinbocanegraalvarado22@gmail.com
Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.
 This document is a property of: Kevin Bocanegra Alvarado

Note: This document is copyright protected. It may not be copied, reproduced, used, or
distributed in any way without the written authorization of Lecturio GmbH.

Kevin Bocanegra Alvarado, kevinbocanegraalvarado22@gmail.com

Powered by TCPDF (www.tcpdf.org)
© www.lecturio.com | This document is protected by copyright.