Stain Chemistry and Technology

Dr Vivien Rolfe
De Montfort University
This is an Open
Educational Resource
(OER) that is globally available
on the web
Creative Commons BY SA
 Histology
is the microscopic study of
normal tissue
 Histopathology is the microscopic study
of diseased tissue
 Cytology is the………………………..
 Cytopathology is the…………………….
 The
light microscope is used to examine
histology slides. You should be familiar
with the parts, operation and principles
of the microscope.
 Use our microscopy OERs to learn how to
use the equipment:
To visualise cell structures and the
chemical nature of tissues.
By Dey on Flickr CC BY NC SA
A dye is a coloured compound that binds to a substrate.
It comprises of the chromogen (colour) and
auxochrome (substrate binding component).
 Dyes
are arranged in 16 groups:
• Basic dyes – coloured cations bind to anionic
substrate e.g. DNA
Acid dyes – coloured anions bind to cationic
substrate e.g. cytoplasm
Mordant dyes – conjugated to metal salts.
Direct dyes
Reactive dyes
• (Kiernan 2007, Histological and Histochemical
The most widely used stain around the world.
Known as the “international stain”.
A simple histological stain with two colours.
Kurt Stüber, Wikimedia Commons, Flickr, CC BY SA
From the bark of the logwood tree Haematoxylum campechianum
 Most
staining is a simple nuclear stain
and a counterstain.
 In most staining protocols, lowest
molecular weight dyes are applied first
(e.g. haematoxylin), differentiated, and
then larger counterstains are applied.
 “Haematoxylin” is a mordant (metal
complexing) dye; the cationic / basic
dye-metal complex binds to nucleic acids
in DNA.
 Haematoxylin
is extracted and oxidised to
haematein. When it complexes with metal it
is more rightly called haemalum.
 It should rightly be the haemalum and eosin
 Aluminium-haematein (e.g. Mayer’s) is
colourised in alkaline tap water (e.g. Scott’s)
which is called blueing, simply to get the
desired final colour.
 Iron-haematein is a deeper blue-black and
is differentiated in stronger acids (e.g.
protocols that use Weigert’s haematoxylin).
Fluorescein, Benjah-bmm27,
Wikimedia Commons
Synthesised from fluorescein, a synthetic organic compound
Used in cosmetics
 Anionic
/acid countertain.
 Eosin Y (a product of fluorescein) binds
to amino acids and most cellular
components, but not for example, larger
molecular structures such as collagen
which are also anionic.
 Stains pink/red.
 Dewax, take sections to water
 Apply haematoxylin
 Rinse
 Differentiate in acid alcohol
(removes dye from cytoplasm
leaving crisp nuclei)
 Rinse
 Blue
 Rinse
 Apply eosin (alcohol based dye)
 Rinse in alcohol
 Dehydrate and clear
Will vary on the tissue makeup.
Cytoplasm-rich muscle will look very pink.
Nuclei-dense tissues e.g. glands look more purple.
Histological and Histochemical Methods. 4th Edition. By JA
Kiernan. 2007. Scion publishing. Available:
Histopathology: Fundamentals of Biomedical Science. By G
Orchard and B Nation. 2012. Oxford University Press.
Laboratory skills open educational resources. De Montfort
University. Available:
Haematoxylin and eosin videos and open educational
resources. De Montfort University. Available:
Dr Vivien Rolfe
De Montfort University
This is an Open
Educational Resource
(OER) that is globally available
on the web
Creative Commons BY SA

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