Theory of Staining Flashcards
What is the term used in light microscopy to refer to the stainable substance in the nucleus?
Chromatin
What does heterochromatin refer to?
Stainable condensed regions of chromosomes that stain intensely basophilic
What is euchromatin characterized by?
Unstainable chromatin representing genetically active DNA, appearing as clear spaces
Which staining method demonstrates intense staining in lymphocytes?
Hemalum Staining
What is the primary physical factor influencing staining reactions?
Most reactions include physical and chemical factors, except lipid staining which is strictly physical
What does dye adsorption refer to in the context of staining?
Attraction of minute particles from solution by the surface of certain tissue components
Define capillary staining.
The ‘taking in’ or ‘sucking up’ of the dye into the minute tubules of tissue
What is adsorption staining also known as?
Electrical theory of staining
Fill in the blank: Selective solubility theory is based on the dye being more soluble in the substance being stained than in the _______.
solvent in which it is dissolved
What does permeability/porosity refer to in staining?
The size of the protein mesh that captures and holds a given dye molecule
What type of bonding occurs when dye and tissue are attracted to each other due to different charges?
Ionic/Electrostatic Bonding
What characterizes covalent bonding in staining?
Occurs when atoms share electrons, forming strong bonds
What is the role of hydrogen bonding in staining?
Occurs when covalently bonded hydrogen is attracted to electronegative atoms
What is hydrophobic bonding in staining?
Holds dyes in tissues by the exclusion of water from hydrophobic groups
What appearance do interphase nuclei typically show when stained by H&E?
Discrete, membrane-bound masses that stain basophilic
What is an accentuator in staining?
A substance that increases the staining power of a dye without forming a link to the tissue
What is the function of a mordant in staining?
Improves binding of tissue elements with the dye by forming a link
What is ripening in the context of hematoxylin staining?
The oxidation product of hematoxylin called hematein during a conversion process
True or False: Basophilic tissues attract basic dyes.
True
True or False: Acidophilic tissues attract acid dyes.
True
Define chromogen.
A compound containing a colour-bearing group or chromophore
What is a chromophore?
The chemical grouping that bestows colour on a compound
What is an auxochrome?
A group of atoms that enables a chromogen to form salts with tissue groups
What distinguishes a cationic dye?
It has a positive charge and is often used for nuclear stains
What characterizes anionic dyes?
They are negatively charged and often used to stain proteins in cytoplasm
What is a ‘lake’ in staining?
A coloured complex formed by the union of dye and mordant
What are the two essential qualities of every dye?
- It must be a coloured substance
- It must attach itself to the tissue
What factors affect dye binding?
- Temperature
- pH
- Concentration
- Time
- Tissue Preparation
What is progressive staining?
A technique where different elements are coloured in sequence until desired intensity is reached
What is regressive staining?
A technique where tissue is over-stained and then differentiated to leave only the desired element stained
What is direct staining?
Staining tissues directly in a simple aqueous or alcoholic dye solution
What is indirect staining?
Staining that requires a mordant before satisfactory combination with tissues takes place
What is argyrophilic reaction in staining?
Reactions where certain tissue elements have a natural affinity for silver nitrate
What is argentaffin reaction?
Use of phenolic groups that can reduce silver salts to stain tissue components directly
What is the result of reducing a bound colourless silver salt like silver nitrate?
Forms a black, reduced silver compound
Reduced by substances such as hydroquinone or formalin
What are Argentaffin Reactions?
Use of phenolic groups capable of reducing silver salts
Tissue elements that can reduce silver ions in solution stain it black or brown
What characterizes argentaffin positive tissues?
The tissue component itself reduces the silver salt directly
Forms visible metallic silver in the tissue without a separate reducing agent
Give an example of an argentaffin material.
Melanin
What happens to carbohydrate-containing tissues when treated with oxidants?
They form aldehydes that can reduce methenamine and silver nitrate
Requires appropriate conditions of temperature and pH
What do histochemical reactions involve?
Reaction between a colourless solution and a specific tissue component
Produces a colour or opaque reaction without needing a dye
True or False: Leuco dyes can become re-colourized by oxidation.
True
What is the purpose of differentiation in regressive staining methods?
Removes stain from sites with less affinity while retaining strong staining in other structures
What is acid differentiation?
Uses dilute solutions of inorganic acids like HCl or acetic acid in solvents
Acts by dissolving dye that has not firmly attached
How do basic dyes differentiate?
By weakly acidic solutions
How do acid dyes differentiate?
By weakly alkaline solutions
What is mordant differentiation?
Uses a weak solution of the mordant used in the original stain
Allows dye to leave tissue and combine with free mordant in solution
What must be monitored during the differentiation process?
Ensure structures that must be stained remain stained with a colourless background
What is solvent differentiation?
A process that depends on dye binding strength, bond type, and solvent volume
Easier to control and can be employed slowly
What does oxidizing differentiation do?
Oxidizes (bleaches) the dye to a colourless substance
Mild oxidizing agents are used for this process
List some solutions used in oxidizing differentiation.
- Potassium ferricyanide
- Potassium permanganate
- Chromic acid
- Picric acid
- Potassium dichromate
What are natural dyes?
Dyes obtained from natural sources like insects and plants
Largely replaced with synthetic dyes
What is Carmine?
A deep red dye extracted from the female cochineal bug
Usually treated with alum or iron to increase its affinity for tissue
What is Hematoxylin derived from?
The heartwood of the logwood tree (Hematoxylum campechianum)
One of the most important natural dyes in biological staining
What is Orcein?
A vegetable dye obtained from certain lichens
Best known as a specific stain for elastic tissue
What is Saffron used for?
Occasionally used in trichrome methods
Limited in use due to high cost
What topic will be covered next week?
Hematoxylin and Eosin Staining and troubleshooting H and E staining
