Staining MELS PDF

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STAINING TECHNIQUES HEMATOXYLIN AND EOSIN What is a stain? Chemical substances used to enhance visibility for microscopic studies Why do we stain? Why are stains and dyes coloured? Why do components stain and others don’t How do we stain? - treating tissues Purpose of stains - To identify tissue - To Observe general morphology - To Demonstrate microorganisms - To Demonstrate particular tissue structures - To Demonstrate pigments - To establish the presence or absence of disease processes (pathologic process). - To enhance visibility - In medical industry = To make a diagnosis COMMONLY USED STAINS 1) H&E 2) Grams 3) Romanosky stain 4) PAP Theory of staining 1. Physical theory - solubility of the dye. 2. Chemical theory - reaction that takes place between the dye and tissue What do they depend on ?

then used this for biological. The dyes that appear colour have the ability to absorb light, the wavelength that don’t get observed are the colours that we can physically see Two types, basic and acidic dye Basic dye—possess a positive charge Acidic dye—possess a negative charge Can be classified into two groups - Natural dyes - Artificial Examples of dyes - Natural - Carmine - bug - Orcein - lichen - Hematoxylin - from a tree, bark ripens. Artificial or synthetic Derived from hydrocarbon benzene 3 Types of Staining 1. Simple Staining- (shapes and arrangements) 2. Differential Staining - (Gram reactions) 3. Special Staining - (Capsule, flagella, spores) How do stains stain? 1. Direct staining 2. Indirect staining - Immunohistochemical method - Metal impregnation technique Direct staining involves bonding between molecules > Probably the most important is ionic bonding where positively and negatively charged ions attract. This is especially seen between charged carboxyl groups and charged amino groups (found in both dyes and tissues) e.g. of ionic bonding is seen in: Where organism is stained and background is left unchanged. Such as in dry film methods. Smears.

Indirect staining Dyes used to be used in a textile industry, they

This includes those stains which need a mordant to link the dye molecule with the tissue component, forming a “lake”. For e.g. with hematoxylin staining Intermediate step for the dye to stain. Needs a middle ground / components added in order for the dyhe to bind with the tissue.

This is due to the structure and composition of the tissue. For example: * haematoxylin is a cationic basic dye, so has an affinity for the anionic acidic nucleic acids in the nucleus. - the blue sections * The hydrophobic molecules of the oil red O dye are attracted to fats and lipids.

e.g.: Immunohistochemical method of staining This method involves the binding of antibodies to antigen with the help of detection agents to visualize the site of interest as a coloured product. Needs a link between

All because of Mordants and Ligands (what the mordants bind to) Chelates (what then is formed

e.g.: method of demonstrating bacteria with an acid dye such as nigrosine. The bacteria remain colorless against a coloured background. Metal impregnation technique. Metallic silver is precipitated within the tissues by reducing the silver salts Tissue may also been stained by: 1. Selective solubility; Where the dye molecule is more soluble in the tissue component than in it’s diluent. 2. Production of an insoluble chemical substance-A histochemical reaction takes place between the substance to be demonstrated and the dye molecule, resulting in an insoluble coloured compound. e.g. Perl’s reaction Iron

LIGANDS - The ion molecule which combines with the metal ion mordan) is called a ligand - Ligands having 2 or more available atoms able to bond with the same metal ion result in forming a stable ring structure or complex called a chelate. - Chelate: a compound containing a ligand (typically organic) bonded to a central metal atom at two or more points.

Why do stains stay in the tissue? 1. Because the stain has a higher affinity for the tissue than the surrounding fluid. 2. Because the product formed by the interaction between stain and tissue is insoluble. 3. Because the dye dissolves slowly in the surrounding fluid. Each dye has a liking. Why do some tissue components stain and not others?

HAEMATOXYLIN 1- digested in alcohol 2- develops a brown colour 3- this is oxidise to become active dye 4- oxidised by sun, next day is oxidised/ripened with chemicals - Nuclear staining is achieved using a mordant Staining methods- H&E 2 methods:

1. ProgressiveTissues are stained until the desired colour is reached. 2. RegressiveTissues are over stained then the dye is selectively removed until the desired level is reached. (Differentiation) Regressive stain Differentiation is the removal of excess dye using acid alcohol, using acetone for gram stain, this is only required in regressive stain.

Refer to your laboratory manual for recipe H&E is a charge- based routinely used stain. Haematoxylin stains acidic molecules shades of blue or purple. Eosin stains basic materials shades of pink. H&E stains are universally used for establishing a histological diagnosis. Acidophil cell- nuclei - are stained purple Basophils cell- cytoplasm are stained pink Why do we counterstain? To enhance the primary stain and provide contrast. Practicalities of staining sections - Can run risks of contamination must work in a safe clean environment - Leaving slides out with cause the tissues to dry out - When applying a stain you should carefully wash of the previously added reagent. Staining considerations - Controls - SOP - Availability - Know safety hazards MSDS - Check reagent expiry dates - Available equipment - Spillage kits Mounting media - Aqueous mountants - Xylol based - Buffer based - close proximity to xylol

Bluing-done in alkaline water. Is the process of shifting the colour range to blue which provides a much better contrast to the pink background. Nuclei will be stained purple- colour of the acid dye. E.g.: Scotts tap water- bluing agent in H&E –

Reagent preps Carefully follow instructions Clearly label bottles with relevant details, e.g.. -Name of stain -Date prepared -Lot/Batch number -Prepared by whom (Staff name/initial) -Expiry date - Hazard symbol Store prepared stain in appropriate location, e.g.. Stain shelf, fridge, dark cupboard, solvent

cupboard Staining Terminologies Basophilic- The entities stainable with basic dye (possess +ve charge) & are substances which are usually acidic in nature. Acidophilic- The entities stainable with acidic dye (possess –ve charge) & are substances which are usually basic in nature. Argyrophilic- The entities stainable with silver nitrate solution. E.g. AgNOR for spirochetes Argentaffin- The entities stainable with silver nitrate solutions without chemical reduction procedures e.g.: Melanin in skin Metachromatic -The entities will stain in a colour or hue different from that of staining solution itself. e.g.: Toluene Blue for mast cells different colours coming in from different wavelengths...