Biological Staining Principles and Techniques

Biological Staining: Terms and Principles

• Biological staining is used in histotechnology and cytology to make tissue components visible.
• Staining procedures in histotechnology and cytology include preparing stains, stock solutions, and working solutions. Reagents and solutions should be managed properly, and appropriate glassware and equipment should be used.
• Dyes, chromophores, chromogens and auxochromes are important components in staining procedures.
• The Biological Staining Commission plays a role in the classification of dyes.
• Dye classification systems and examples of natural and synthetic dyes are key components in staining.
• Staining theories, techniques, direct staining, indirect staining, mordants, accentuators, trapping agents, vital staining, and the physical and chemical theories of staining are important principles.
• Metallic impregnation, metachromatic staining, and fluorescent staining are additional staining techniques.
• Staining terminology is essential in understanding staining protocols.
• Stain preparation may involve commercial preparation or in-house preparation of stains. Quality control is maintained by testing new and existing reagents/stains

Staining Solutions

• Some stains and reagents are purchased commercially.
• Other stains and reagents are prepared in-house.
• Stock and working solutions are prepared following standardized protocols or recipes.
• Quality control for reagents/stains is maintained by testing the materials before use and during each staining process.

Storage and Handling

• All chemicals must be properly labeled, including preparation date, expiration date, and WHMIS labels.
• Chemicals should be stored according to their specific requirements (e.g., flammables, explosives, oxidizers).
• Chemicals should be disposed of according to guidelines (e.g., solvents).
• Proper ventilation and PPE should be used when handling staining solutions.

Natural and Synthetic Dyes

• Natural dyes are derived from plants, animals, and insects.
• Synthetic dyes are derived from benzene.

Dye classifications

• Classification of dyes includes natural and synthetic dyes, chromophores, acidic dyes, basic dyes, and neutral dyes.

Making things obvious or visible

• Dyes or stains increase the visibility of the objects.
• Chemical reactions in combination with dyes may produce a visible result.
• Metallic deposits can create visible differences in objects.

Dye Molecule

• Chromophores are the colored groups in a dye molecule.
• Auxochromes are groups that increase the water solubility of dye.
• Chromogens are parts of molecules that add color.

Chromogens and Auxochromes

• Chromogens are the colored parts of a dye molecule.
• Auxochromes are side groups in a dye molecule that enhance the ability of dye to bind to tissue elements.
• Auxochrome ionizes the chromogen to give it a charge, allowing it to bind to substances.

Part of a dye molecule

• Auxochromes are groups needed for electrochemical bonding of the dye to the substance.

What is a color index number?

• A five-digit number that identifies a dye's precise chemical and structural composition.
• The number helps avoid confusion caused by dyes having multiple names.

How does pH affect staining?

• Staining relies on attraction between dye and tissue components based on charge differences (acidic to basic).
• The charge on proteins in tissue depends on the pH of the surrounding environment.
• An optimal pH exists where the tissue is precisely stained, avoiding overstaining or understaining.

How does temperature affect staining?

• Chemical reactions, including staining, occur faster at higher temperatures.
• Temperatures above 37°C cause tissue swelling and increased penetration, aiding in faster staining of certain types of tissue.

How will dye concentration affect staining?

• Increased dye concentration speeds up the staining reaction, but only up to the point all binding sites are occupied.

What is a true dye?

• A true dye is an organic compound containing chromophore and auxochromic groups.

Theories of staining

• Physical (absorption and preferential solubility) and chemical (dye with tissue, histochemical reactions, and impregnation) theories play a role in staining.

Physical considerations in staining

• Larger dye particles diffuse slower than smaller ones.
• Higher tissue porosity allows staining to be easier and faster
• Dye concentration at the surface and interface of the solution with the tissue can contribute to staining intensity.

Preferential (selective) solubility

• Dyes that are more soluble in tissue components than in solvents will migrate from the outside to the tissue.

What is a lysochrome?

• A colored substance that stains substances in which it is soluble without auxochromes (e.g., Oil Red O, and Sudan Black).

What is a true histochemical reaction?

• A chemical reaction between tissue elements and reagents that leads to the localized production of coloured compounds. (e.g., Prussian blue).

What is an isoelectric point of protein?

• Most proteins have an isoelectric point near pH 6.
• At this point, proteins have a balance of charges preventing strong attraction to cationic or anionic dyes.
• The pH of solution changes the charge on proteins.

What is a mordant?

• A mordant is a substance, often a metal, that provides a link between a dye and a tissue.
• When a mordant is combined with a dye, the composite is called a "dye lake".

Metallic impregnation

• A non-dye method that uses metallic salts to visualize fine structures.
• Some types include Argentaffin (reduction) and Argyrophil (external reduction).

Are cytoplasmic proteins basophilic or acidophilic?

• Cytoplasmic proteins are acidophilic at pH 6 since they are cationic, making them attracted to anionic dyes.

Progressive vs. Regressive Staining

• Progressive staining involves staining until the desired intensity is achieved.
• In regressive staining, tissue is overstained then the substance is removed until the desired intensity is achieved. Microscopic control is essential.

Comparing progressive and regressive hematoxylin

• Progressive hematoxylin procedures are generally less concentrated and work slower to avoid overshooting the endpoint.
• Regressive hematoxylin solutions are more concentrated and some may overstain within a minute.

Amount of bonding affected by

• Ionic strength, dye concentration, tissue fixation, temperature, pH of the solution, and thickness of tissue can affect bonding amounts.

What is the pivotal reagent for IHC?

• An antibody, usually monoclonal, is the pivotal reagent.

What are some applications of IHC

• IHC can help give better cancer diagnoses, aid in selecting appropriate cancer treatments, diagnose viral infections, and identify APUD cells.

Explore the world of biological staining and its crucial role in histotechnology and cytology. This quiz covers staining procedures, key components like dyes and chromophores, and various techniques such as vital staining and metallic impregnation. Test your understanding of staining terminology and principles.