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:  RNdt3JM

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). Chromogen Auxochrome Substrate

 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 Others (Kiernan 2007, Histological and Histochemical Methods).

The most widely used stain around the world. Known as the “international stain”. A simple histological stain with two colours.

Haematoxylin From the bark of the logwood tree Haematoxylin From the bark of the logwood tree Haematoxylum campechianum Flickr, CC BY SA Kurt Stüber, Wikimedia Commons

 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 stain.  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).

Eosin Synthesised from fluorescein, a synthetic organic compound Used in cosmetics Fluorescein, Benjah-bmm27, Wikimedia Commons

 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. 4 th Edition. By JA Kiernan Scion publishing. Available:  Histopathology: Fundamentals of Biomedical Science. By G Orchard and B Nation Oxford University Press. Available:  Laboratory skills open educational resources. De Montfort University. Available:  Haematoxylin and eosin videos and open educational resources. De Montfort University. Available: ml

Dr Vivien Rolfe De Montfort University This is an Open Educational Resource (OER) that is globally available on the web Creative Commons BY SA