Dye(8/12)

Anmerkungen:

• Basic definition: 1. Dyes are aromatic compound-can dissociate in water to form ions(cation and anion)

1. Dye consitution
   1. Chromophore

      Anmerkungen:

      • give color to the dye
      1. Properties

         Anmerkungen:

         • 1. atomic configurations that contain delocalized electrons: e.g nitrogen, oxygen,carbon, sulphur:they have alternate single or double bonds
         • the most important chromophore:quinoid arrangement of the benzene ring
         1. **quinoid rings

            Anmerkungen:

            • 1. a ring structure with two points for chromophore attachment why color produced?? 1. adding delocalised electrons to the system at one point, extend the atoms involved in the delocalization at the other
            • cause dramatic shift in the wavelengths which these compounds absorb
      2. known as chromogen

         Anmerkungen:

         • containing chromophore radical but not an auxochrome
   2. Auxochrome

      Anmerkungen:

      • gives a charged end group to the dye:
      1. Mechanism

         Anmerkungen:

         • 1. additional auxochromes: turn a colored compound into a dye--by forming ionic bound(with an oppositely charged group in the tissue)
         • e.g. ionizable OH groups turn trinitrobenzene to trinitrophenol picric acid
   3. fluorochrome

      Anmerkungen:

      • dyes with usual chromophores and auxochromes: can alter UV or short wavelength light into visible light
      • example: 1.Auramine O for AFB(acid fast bacilli) 2.Acridine orange for DNA/RNA 3.Thioflavine T for amyloid
2. modification for the dye

   Anmerkungen:

   • using modifiers!!
   1. affect color
      1. e.g. CH3,C2H5 or acryl groups
         1. replacing H

            Anmerkungen:

            • replacing H on the benzene ring or basic auxochromes: gives blurer shade to the dye
   2. affect properties of the dye
      1. e.g. sulfonation

         Anmerkungen:

         • it is a treatment with sulfuric acid to produce different dye(e.g. acid fuchsin)
         1. such as acid fuscin

            Anmerkungen:

            • it is a mixture of homologues basic fuchsin + sulfonic groups
3. Classifications
   1. 1.simple dye
      1. Basic/cationic dye
         1. properties

            Anmerkungen:

            • 1.cationic:stain anionic or acidic material(basophilic)(e.g. sulphated carbohydrates,phosphates in nucleic acids)
            • acidic substances:sulphate, chlorides or acetate
         2. usages

            Anmerkungen:

            • 1.used as nuclear stain 2.other acidic tissue components: a.DNA/RNA b.Phospholipids c.mareix of cartilage d.mucinous secretion
      2. Acid / anionic dye
         1. properties

            Anmerkungen:

            • 1.anionic 2.derived from coloured acids 3.stain cationic/basic groups in tissue(e.g. amino group)
         2. usages

            Anmerkungen:

            • 1. use to stain cytoplasm / connective tissue**(acidophilic substances) 2.other substances: sodium, potassium, calcium, ammonium
            • basic tissue constitutes: 1.collagen 2.cytoplasm of RBC 3.eosinophilic granules
      3. Amphoteric dyes

         Anmerkungen:

         • have both anionic and cationic groups on the same ion: -become basic dye: at pH below it iso-electric point(IEP) -become acid dye :at pH above IEP
   2. 4. mordant dyes or indirect dye+direct dyes
      1. direct dyes

         Anmerkungen:

         • dye have direct affinity for tissue -attachment can affected by tissue permeability and density
         1. attach tissue by electronic bonds

            Anmerkungen:

            • 1.anionic dye to cationic tissue components 2.cationic dye to anionic tissue components
      2. mordant dyes and indirect dyes

         Anmerkungen:

         • 1.dyes have little affinity for tissue 2.require intermediate binding agent(mordant): for dye-tissue reaction
   3. 2. compound or neutral dye

      Anmerkungen:

      • 1. compound of acid and basic dyes: both basic ions and acid ions are coloured 
      1. usages

         Anmerkungen:

         • 1. stain both nucleus and cytoplasm from a single dye bath
         • e.g. methylene blue eosinate(prepared by romanowsky:staining blood cells) expected result:3 color are produced due to partial dissociation. 1. neutaral stain:puplish stain nucleus 2.colored base:blue, stain basophils 3.coloured acid:red, stain eosinophils
   4. 3. natural and synthetic dye

      Anmerkungen:

      • 1.extracted from natural sources 2.largely replaced by synthetic dyes
      1. a. natural dyes

         Anmerkungen:

         • still in use: 1.hematoxylin:from logwood 2.carmine:from cochineal(female coccus cacti insert)
         • 3. orcein:from lichen 4. saffron:stigma of crocus sativus
      2. b. synthetic dyes

         Anmerkungen:

         • 1.reliable 2.cheaper 3.supplied more readily
         • two examples 1.coal tar dye:from hydrocarbons obtained by distillation of coal tar 2.aniline dye: intermediate product between dye and benzene
   5. 5.metachromatic dye and metachromasia

      Anmerkungen:

      • metachromatic dye: stain tissue component a different color to a dye solution
      1. application

         Anmerkungen:

         • e.g. Toluidine stains nucleui a deep blue color but mast cell granules with a pink color -the usual blue stain=orthochromasia -the color shift=metachromasia
      2. principle for metachromasia

         Anmerkungen:

         • 1.the colour shift: from a blue to violet dye to yellow or red staining: colour absorption shift to shorter wavelength and only the longer wavelength to be seen.
         1. represent polymerization of the dye

            Anmerkungen:

            • the greater the degree of polymerization=stronger metachromasia( affected by dehydration and clearing)
      3. **chromotrope

         Anmerkungen:

         • 1. alter the colour of metachromatic dye(e.g. sulphated mucins) -metachromasia is exhibit when the dye is bind to certain substance called chromotrope
4. Non-dye constitution of staining solution
   1. mordants
      1. properties

         Anmerkungen:

         • 1. act as a link between the dye and the tissue common- a salt of a metal or an alum e.g. Cr, Al, Fe,K, Pb,tungsten
      2. mechanisms

         Anmerkungen:

         • using dative covalency
         1. dative bonds on the dye

            Anmerkungen:

            • The group on the dye forming dative-bond: 1.oxygen containing: in phenols, carboxyl, quinones 2.nitrogen containing: in amine, azo, nitro groups
            1. forming dye lakes(complex of dye + mordant)

               Anmerkungen:

               • how it forms:1.the covalent bonds form between hydroxyl oxygen and metal 2.coodinated bonds form between the doubled bond oxygen and the metal
               1. attached to the tissue

                  Anmerkungen:

                  • same mechanism as the formation of the dye lakes
      3. classifications
         1. 1.metachrome

            Anmerkungen:

            • 1. staining is done with dye lake(alum-hx)
         2. 2. onchrome

            Anmerkungen:

            • 1. mordant is applied first, followed by the dye e.g. Heidenhain's iron hx
         3. 3. afterchrome

            Anmerkungen:

            • Dye is applied first and mordant add afterward.
      4. usages
         1. progressive

            Anmerkungen:

            • with the dye being applied to the section, until desired color is reached
         2. regressive(used commonly)

            Anmerkungen:

            • 1.overstain the tissue to darker than is needed 2. differentiation to obtain desired color by removing excess stain(dye-lake)
            • problems 1. dyes are rarely specific 2.will not only stain the structure, can also stain the background
            1. differentiation is required

               Anmerkungen:

               • 1. strong acids: e.g. hydrochloric acid(often in alcoholic solution): MOA: disrupt the tissue-mordant bonds rather than mordant-dye bonds 
               • 2.excess mordants: e.g. iron alum in Heidenhain's Hx MOA: displace the  dye lake and replaced by mordants with no attached dye
   2. trapping agents

      Anmerkungen:

      • e.g. use iodine to trap the violet dye in stain
      1. features

         Anmerkungen:

         • 1. applied after dye
         • 1. form large aggregate with the dye 2.resulting in dye precipitating(difficult to remove) in the tissue
   3. accentuators and accelerators
      1. properties of accentuator

         Anmerkungen:

         • e.g. phenol in carbon fuchsin
         1. increase specificity and staining power of the dye
         2. increased affinity of the tissue
         3. not involved in the staining reaction
         4. control pH
      2. properties of accelerators

         Anmerkungen:

         • e.g. acetic acid in eosin
         1. speed up the reaction

            Anmerkungen:

            • between tissue and stains
5. Dye analysis
   1. spectrophotometry
   2. chromatography
   3. electrophoresis
   4. chemical titration