Topic 8: Staining Theory

What is staining

Visual labelling of an area of interest within cells or tissues. It gives a characteristic color to a substance or structure.

What is a dye

Any colored substance that has an affinity for a particular substrate

How are synthetic dyes create

Aromatic hydrocarbons which are substituted with various functional groups to absorb different wavelengths of light, which are perceived as different colors.

The 4 examples of natural dyes

hematoxylin (from the logwood tree, Haematoxylum campechianum)

carmine (from the female cochineal insect, Dactylopius coccus)

orcein (found in several lichens, especially Roccella tinctoria)

saffron (from the stigma of the saffron crocus, Crocus sativus)

What is a chromophore

A molecular structure that absorbs visible wavelengths of light, which makes it appear as a particular color.

Why do some atomic structures absorb light compared to others

The more “spread out” (delocalized) the electrons are in an organic molecule, the more likely it is to absorb visible wavelengths of light. Double- or triple-bonds are more delocalized than single-bonds; double- or triple-bonds alternating with single bonds in series are even further delocalized.

What is a chromogen

The part of the dye that contains the chromophore(s) (think color generator)

What is an auxochrome

ionized molecular structures which allow the dye to attaching to the tissue, usually by forming salts with tissue anions or cations

A dye which contains an anionic auxochrome is a(n) ___ dye

Anionic (acid)

A dye which contains a cationic auxochrome is a ___ dye

Cationic (basic)

Examples of anionic auxochromes

Carboxyl (-COOH), Hydroxyl (-OH), Sulphonic (-SO3H) groups. These groups ionize and bind to positively charged tissue components

Common binding sites for anionic dyes

Amino acids lysine, histidine, arginine of many proteins.

Common anionic dyes

Eosin, Orange G, Biebrich scarlet, acid fuchsin

Examples of cationic auxochromes

-NH2. It forms -NH3+ which binds with negatively charged tissue components.

Common binding sites for cationic auxochromes

Phosphate groups of nucleic acids and sulfated polysaccharides (cartilage, basophil granules, and acid mucins)

Common cationic dyes

Hematoxylin, crystal violet, methylene blue, basic fuchsin

Is trinitrobenzene a dye?

No, as it does not contain any auxochromes

Is pictic acid a dye?

Yes. It has a hydroxyl group which can ionize and react with positvely charged components (anionic dye)

What is a mordant

A reagent that links the dye to the tissue.

How do mordants help link dye to tissues

Mordants are metallic salts (usually aluminum or iron) that form a complex with certain dyes. It is theorized that the positive charge allows it to bind to anionic tissue components, as well as form chelates in some circumstances

A dye combined with a mordant is called___

Dye lake

What is an accenuator, and what are some examples

Accenuators increase the intesnity of the dye.

Examples are acids added to anionic dyes, or bases added to cationic dyes

What is a neutral dye

A staining solution that contains a mixture of acidic and basic dyes

Composition of Romanowsky stains

Oxidized methylene blue (forming azure A, B and other dyes) with eosin (acidic dye)

What is progressive staining

slides are placed in the desired stain for a pre-determined amount of time, in order to stain to the desired intensity. No differentiator is used in this method. 

What is regressive staining

 The tissue is overstained , then decolourized/differentiated until the desired element is well-demonstrated and less desirable tissue elements are left unstained.

What is differentiation

AKA decolorization.  The removal of excess stain. Usually an acid is used (H&E), although using excess mordant is also effective (Verhoeff’s elastin).

What is metahchromasia

 Tissue elements are said to be metachromatic when they stain a different colour than that of the staining solution

What causes metachromasia in tissues

 certain cationic dyes (toluidine blue, methylene blue, crystal violet) form dye aggregates (or possibly polymerize) when staining substrates that contain many negatively-charged sites in close proximity. Usually, the colour will change from blue to purple or from purple to red.

Examples of metachromatic substances

Mast cells, mucins, amyloid

What is polychromasia

Different shades of a stain are present on the tissue. Due to tissue elements selectively binding to different dye components in the solution

Chemical staining mexhanism

Chemical reactions result in the formation of a bond. Some of these bonds are more permanent than others

Covalent bonding stain mechanism

A strong bond formed between sharing electrons between atoms

Examples of covalent bonding stains

Schiff reagent in PAS stain, Feulgen reaction, iron-mordanted hematoxylin staining

Ionic bonding (electrostatic binding, salt linkage) staining mechanism

Attraction between unlike charges. Ionic bonds are weaker than covalent, but stronger than hydrgen bonds. Most common mechanism of staining

Examples of ionic bonding stains

Alum-hematoxylin, eosin, alcian blue, crystal violet, many others

Hydrogen bonding staining mechanism

involves attraction between hydrogen atoms of one molecule and nitrogen or oxygen atoms of another molecule.

What is the issue with hydrogen bonding stains

Water will interfere withhydrogen bonding as it extensively hydrogen binds with itself

Examples of stains with hydrogen bonding

Elastin stains and Congo red (amyloid)

Physical staining mechanism

Some stains function by virtue of their physical properties, particularly size and solubility.

what stains use selective solubility

Lipid demonstrating stains (Oil red O, Sudan Black)

Selective solubility staining mechanism

The dye is only slightly soluble in a chosen solvent, and readily soluble in fats. This results in dye molecules migrating from its solvent into the lipids of the tissue section

Porosity staining mechanism

Several dye solutions are used, each with varying sizes of dye molecules. The smallest dye molecules are applied first, which quickly fill porous structures within the tissue. Larger dye molecules are then added, which “bump” the smaller dye molecules out of the larger pores. Once the dye molecules are “seated” in the pores, they are held in place by ionic bonds.

Examples of porosity stains

Masson trichrome, and other connective tissue stains

What is Van der Waal’s forces

transient intermolecular attractions and dispersions that occur due to molecular polarity (dipole-dipole interactions).  As polar molecules interact in solution, the positive dipole of one molecule may be attracted to the negative dipole of another adjacent molecule. When molecules are held in very close proximity, these forces may become significant.

Stains that use Van der Waals forces to their advantage

Congo red staining, and some elastin stains

Histochemical staining mechanism

does not employ a dye or stain. By utilizing a chemical reaction that forms a coloured end product, specific tissue elements can be selectively demonstrated.

Example of a histochemical mechanism stain

Perl’s Prussian blue stain, where ferric iron (the major component of hemosiderin) reacts with an acid-ferrocyanide solution forming a bright blue insoluble product (Prussian blue). Note that any ferric iron present will stain, not only the ferric iron in hemosiderin.

Silver staining methods (metallic impregnation)

silver nitrate solutions are easily reduced, forming metallic silver deposits. Therefore, they can be used to “stain” substances that are capable of reducing silver.

3 categories of silver stains

Argentaffin, Agyrophil, Metallic substitution

Argentaffin substance

ny substance that can bind silver from solution, and reduce it to its metallic (visible) form

Examples of argentaffin substances

Melanin, Argentaffin granules (within enterochromaffin cells), formalin pigment, some neurosecretory granules

Argyrophillic substances

tissue components are capable of binding silver from solution, but have no ability to reduce the silver to its metallic form. In order to visualize the bound silver, a chemical reducing agent must be applied.

Examples of argyrophillic substances

Urates and argyrophil granules. Other substances can be pre-treated to induce argyrophilia, such as reticular fibers.

Relationship between argyrophil methods and argentaffin substances

Argyrophil methods will also demonstrate argentaffin substances.

Metallic substitution

A special case of silver staining utilized by the Von Kossa method for calcium

How does metallic substitution work (Von Kossa stain)

Silver will react with the carbonate and phosphate anions of calcium salts, replacing the calcium. Bright light is then used to reduce the silver salt to metallic silver. Any silver “staining” is assumed to be at sites that used to contain calcium. This is an indirect method for calcium, as the calcium itself is not detected.

Von Kossa staining is an indirect calcium stain. What is a direct calcium stain?

Alizarin red S