Module 1: Staining Mechanisms

Question 1

Vital staining

Answer 1

• staining of living tissue by demonstrating cytoplasmic structures through phagocytosis of dye particles (ie; reticulocyte)
• nuclear membrane of living cell is impermeable to dyes
• not applicable to fixed, sectioned tissue

Question 2

Selective/Preferential solubility

Answer 2

• dye is soluble in certain tissues aside from solvent dissolved in (Oil Red O for lipid demonstration)

Question 3

Histochemical techniques

Answer 3

• chemical reagents that react with tissue to produce colored substances
• PBB (ferric iron), PAS (carb), azo coupling reactions (enzymes)

Question 4

Metachromasia

Answer 4

• certain tissues combine with dyes to produce a color different than original dye, a color different from what’s produced
  in the rest of the tissues
• chromotrope is a substances that can alter the color of a metachromatic dye. Usually high MW, made up of large
  anionic molecules containing sulphate, phosphate or carboxylic acid radicals in abundance
• metachromatic dyes are all aniline basic, most belong to thiazine group and are all capable of forming polymers
• acid polysaccharides are main group of tissue chromotropes and occur in amyloid, matrix of cartilage, mast cell
  granules, mucin and connective tissues containing mucins (all anionic and have affinity for basic dyes) – in these
  substances these groups are close enough together to permit secondary bonding or polymerization of bond dye molecules

Question 5

Three categories of metachromasia

Answer 5

Alpha: negative (orthochromatic) - blue (monomers)
Gamma: full- pink to red (polymers) - Toluidine blue, methylene blue and crystal violet
Beta: Intermediate - purple (di and trimmers)

Question 6

Metallic impregnation

Answer 6

• deposit of salts (silver or gold) over selective cells and tissue structures followed by reduction of soluble and colorless
  substance to leave opaque insoluble residue
• ammoniacal silver (reticulin, fungi, melanin and BM) and gold salts (nerve tissue)

Question 7

3 types of metallic impregnation

Answer 7

Argentaffin
Modified argentaffin
Argyrophil

**Not true staining - no chromophore groups, silver deposited on or around but not within, solutions are hetero

Question 8

Argentaffin

Answer 8

tissue component possesses capacity to reduce silver. Easiest. Melanin & enteroendocrine granules

Question 9

Modified Argentaffin

Answer 9

demonstrate fungus and Pneumocystis

Question 10

Argyrophil

Answer 10

tissue components have affinity and can be impregnated with silver, but deposits are on molecular
level (can’t be seen unless reducing agent is applied. Reticulin

Question 11

Metallic substitution

Answer 11

• occurs in demonstration of calcium salts. Ion exchange occurs between calcium and silver nitrate solution – light or
  reducer used to blacken silver

Question 12

Leuko compounds

Answer 12

• colorless compounds occurring due to reduction. Reversible – recolor via oxidization
• Schiff’s reagent prepared from basic fuschin. Not true leuko dye (chromophoric reconfiguration occurs when new
  product is formed). Used to localize particular tissue components if it’s capable of chromophore production within the
  leuko dye which restores the chromophore, establishing insoluble color at site

Question 13

Polychromasia

Answer 13

Polychrome methylene blue
Fluorochromes
Mordanted dyes
Nuclear staining

Question 14

Polychrome methylene blue

Answer 14

• pure but unstable and oxidize easily – allows spontaneous formation of other dyes (methylene blue – Azure A, B and
  methylene violet)
• combined with eosin in prep of Romanowsky stain – stains orthochromatic tissue varying shades of blue, and
  chromotropes in shades of reddish violet

Question 15

Fluorochromes

Answer 15

• absorb UV or blue light and emits a longer wavelength
• usually colorless and are often used to label site of antigen/antibody reactions in immunofluorescent methods
• Thioflavine T and Acridine Orange

Question 16

Mordanted dyes

Answer 16

• some dyes have no affinity for tissue elements unless combined with metallic salt (mordant) which is di or trivalent and
  can form a link between dye and tissue (covalent bonding). Most common are potassium or ammonium aluminul sulphate
  and ferric ammonium sulphate
• dye + mordant = lake
• lake reacts with charged chemical components of tissue (important in nuclear staining)

Question 17

Nuclear staining

Answer 17

• can occur by metal mordant dyes (hematoxylin) or with cationic (+) dyes which link with acidic (-) nucleus (salt linkage)
• alums – class of mordant, double sulphate salts of aluminum, iron and chromium.