Dye(8/12)

Annotations:

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

1. Dye consitution
   1. Chromophore

      Annotations:

      • give color to the dye
      1. Properties

         Annotations:

         • 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

            Annotations:

            • 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

         Annotations:

         • containing chromophore radical but not an auxochrome
   2. Auxochrome

      Annotations:

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

         Annotations:

         • 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

      Annotations:

      • 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

   Annotations:

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

            Annotations:

            • 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

         Annotations:

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

            Annotations:

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

            Annotations:

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

            Annotations:

            • 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

            Annotations:

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

            Annotations:

            • 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

         Annotations:

         • 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

         Annotations:

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

            Annotations:

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

         Annotations:

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

      Annotations:

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

         Annotations:

         • 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

      Annotations:

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

         Annotations:

         • 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

         Annotations:

         • 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

      Annotations:

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

         Annotations:

         • 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

         Annotations:

         • 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

            Annotations:

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

         Annotations:

         • 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

         Annotations:

         • 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

         Annotations:

         • using dative covalency
         1. dative bonds on the dye

            Annotations:

            • 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)

               Annotations:

               • 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

                  Annotations:

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

            Annotations:

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

            Annotations:

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

            Annotations:

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

            Annotations:

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

            Annotations:

            • 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

               Annotations:

               • 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

      Annotations:

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

         Annotations:

         • 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

         Annotations:

         • 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

         Annotations:

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

            Annotations:

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