Dye(8/12)

Dye(8/12)

1. Dye consitution

   • Chromophore
     • give color to the dye
     • Properties
       • 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
       • **quinoid rings
         • 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
     • known as chromogen
       • containing chromophore radical but not an auxochrome
   • Auxochrome
     • gives a charged end group to the dye:
     • Mechanism
       • 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
   • fluorochrome
     • 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

   • using modifiers!!
   • affect color
     • e.g. CH3,C2H5 or acryl groups
       • replacing H
         • replacing H on the benzene ring or basic auxochromes: gives blurer shade to the dye
   • affect properties of the dye
     • e.g. sulfonation
       • it is a treatment with sulfuric acid to produce different dye(e.g. acid fuchsin)
       • such as acid fuscin
         • it is a mixture of homologues basic fuchsin + sulfonic groups

3. Classifications

   • 1.simple dye
     • Basic/cationic dye
       • properties
         • 1.cationic:stain anionic or acidic material(basophilic)(e.g. sulphated carbohydrates,phosphates in nucleic acids)
         • acidic substances:sulphate, chlorides or acetate
       • usages
         • 1.used as nuclear stain 2.other acidic tissue components: a.DNA/RNA b.Phospholipids c.mareix of cartilage d.mucinous secretion
     • Acid / anionic dye
       • properties
         • 1.anionic 2.derived from coloured acids 3.stain cationic/basic groups in tissue(e.g. amino group)
       • usages
         • 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
     • Amphoteric dyes
       • 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
   • 4. mordant dyes or indirect dye+direct dyes
     • direct dyes
       • dye have direct affinity for tissue -attachment can affected by tissue permeability and density
       • attach tissue by electronic bonds
         • 1.anionic dye to cationic tissue components 2.cationic dye to anionic tissue components
     • mordant dyes and indirect dyes
       • 1.dyes have little affinity for tissue 2.require intermediate binding agent(mordant): for dye-tissue reaction
   • 2. compound or neutral dye
     • 1. compound of acid and basic dyes: both basic ions and acid ions are coloured 
     • usages
       • 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
   • 3. natural and synthetic dye
     • 1.extracted from natural sources 2.largely replaced by synthetic dyes
     • a. natural dyes
       • 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
     • b. synthetic dyes
       • 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.metachromatic dye and metachromasia
     • metachromatic dye: stain tissue component a different color to a dye solution
     • application
       • 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
     • principle for metachromasia
       • 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.
       • represent polymerization of the dye
         • the greater the degree of polymerization=stronger metachromasia( affected by dehydration and clearing)
     • **chromotrope
       • 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

   • mordants
     • properties
       • 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
     • mechanisms
       • using dative covalency
       • dative bonds on the dye
         • The group on the dye forming dative-bond: 1.oxygen containing: in phenols, carboxyl, quinones 2.nitrogen containing: in amine, azo, nitro groups
         • forming dye lakes(complex of dye + mordant)
           • 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
           • attached to the tissue
             • same mechanism as the formation of the dye lakes
     • classifications
       • 1.metachrome
         • 1. staining is done with dye lake(alum-hx)
       • 2. onchrome
         • 1. mordant is applied first, followed by the dye e.g. Heidenhain's iron hx
       • 3. afterchrome
         • Dye is applied first and mordant add afterward.
     • usages
       • progressive
         • with the dye being applied to the section, until desired color is reached
       • regressive(used commonly)
         • 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
         • differentiation is required
           • 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
   • trapping agents
     • e.g. use iodine to trap the violet dye in stain
     • features
       • 1. applied after dye
       • 1. form large aggregate with the dye 2.resulting in dye precipitating(difficult to remove) in the tissue
   • accentuators and accelerators
     • properties of accentuator
       • e.g. phenol in carbon fuchsin
       • increase specificity and staining power of the dye
       • increased affinity of the tissue
       • not involved in the staining reaction
       • control pH
     • properties of accelerators
       • e.g. acetic acid in eosin
       • speed up the reaction
         • between tissue and stains

5. Dye analysis

   • spectrophotometry
   • chromatography
   • electrophoresis
   • chemical titration