Lecture 6 Part A: Stain Theory

Chemistry of Dyes

Natural

Synthetic

Most synthetic = organic
-aromatic
-derived from benzene ring

Benzene ring in dyes:
-known as QUINONE RING
-C6H4O2
-oxygen replaces hydrogens in ortho or para positions
-a.k.a QUINOID QUINONOID compounds


CHROMOPHORE

atomic groupings associated with colour

part of dye molecule

e.g: C=C, C=O, C=S, C=N or N=N, N=O, NO2

COLOUR THEORY

More chromophores in compound = more pronounced colour

Physical chemistry suggests colour caused by selective absorption of certain wavelengths of light

CHROMOGENS

Aromatic compounds containing chromophore radicals

Act as solvent dyes

Colours oils, fats + waxes by dissolving them (LYSOCHROME)

AUXOCHROME

chemical group in dye molecule that ionises

enhances action of chromophore

intensifies colour of the dye

type of auxo group will determine action of the dye and to which tissue elements it will bind
-chromogens require auxochromic groups for binding (either electrostatic or covalent depending on tissue end groups)

ACIDIC (-COOH)
-loses H+
-acts as anions
-negatively charged

BASIC (-NH2)
-supply H+
-act as cations
-positively charged

Biological Dyes

Requirements:CASSPRR
-reveal detail sought in the tissue
-colour intensity C
-affinity to tissue A
-chemically reliable
-adequate solubility S
-stability S
-purity P
-consistency for reproducible results R
-resistance to changes in colour intensity R
from light, extreme pH, leaching by mounting medium*

DYE PURITY
-determined by CI (colour index number)
-requires certification by US Biological Staining Commission
-necessary for consistency + quality control

FUNCTION OF STAINING
-differentiate cell cytoplasm from nucleus
-distinguish betw. tissue types
-nature of the dye gives specificity to tissue elements

DYE TO TISSUE BINDING
-chemical + physical forces
-hydrophobic bonding
-Van der Waal's forces
-Coulombic attractions
-hydrogen bonds
-covalent bonds
-rate of penetration (tissue lattice structure vs molecular size of dye)
-rate of uptake/reaction
-rate of loss during differentiation

ORTHOCHROMASIA
-tissue staining the same colour and shade as the one dye BUT of varying intensity
-e.g. Methylene Blue

METACHROMASIA
-tissue staining same and different shades of the one dye
-e.g. Toluidine Blue

POLYCHROMASIA
-tissue staining many colours and shades in a staining solution made up of compound of multiple dyes
e.g. Romanowsky type dyes to stain blood films, Leishman's, May Grünwald Giemsa, Wright's stains

-Binds to form salts with negatively charged tissue elements
-binds to basophilic substances
-e.g. nucleic acids
-stains best at basic pH
-e.g. Methylene Blue

-binds to form salts with positively charged metal ions in or assoc. with tissue elements
-binds to acidophilic substances
-stains best at acidic pH
-e.g. Eosin, Biebrich Scarlet

AMPHOTERIC DYES
-dye BOTH positively + negatively charged groups
-function as acid or base depending on pH of solution
-e.g. Haematein
-basic radicals of acid dyes (NH+) colourless
-acid radicals of basic dyes (COOH-) colourless
-tissue proteins can be amphoteric, charge will vary depending on pH and therefore will bind to either basic or acidic dyes

NEUTRAL DYES
-combination of acid and base dyes in correct proportions will form water-insoluble neutral compounds
-made up as alcoholic solutions
-ionize when solution diluted with H2O
-selectively stain tissues that are oppositely charged
-give polychromasia
-often used in blood films

HAEMATOXYLIN STAIN
-C.I. 75290
-requires mordant to act as 'bridge' betw dye + tissue (a metal ion)
-mordant binding of haemotein molecules = co-ordination complex
-bond stable + fixed when alkaline

METAL ION MORDANTS
-type of metal influences tissue elements that the haemotoxylin stains
-e.g. aluminium (commonly used for H&E), iron (used in H&E when counterstain is acid), tungsten, molybdenum, lead

HAEMATEIN
-oxidised form of haematoxylin
-formed when haematoxyling exposed to oxygen in presence of light
-or done chemically by adding sodium iodate or mercuric oxide
-amphoteric when acidified + has red bias
-when alkaline has blue bias

FORMS OF HAEMATOXYLIN STAINING
-PROGRESSIVE
-does not need differentiaiton
-not overstained
-e.g. Gill's, Mayer's


-REGRESSIVE
-needs differentiation
-oversatined
-e.g. aluminium mordanted, Harris', Erlich's, Heidenhain's

DIFFERENTIATION
-removal of excess dye from over-stained (regressive) tissue
-enhances details of cellular structure
-bond betw haematoxylin dye + mordant acidified w/ acid alcohol which detaches the dye

BLUING
-re-establishes co-ordination complex once differentiation step is completed
-re-attaches dye to tissue section
-tissue needs to be realkalinised by washing with water
-OR use Scott's bluing solution strengthens haematein/mordant bond + precipitates dye to prevent further loss

EOSIN Y DYE
-common counterstain for haematoxylin
-xanthene dye
-tatrabromo derivative of fluorescin
-Eosin Y most widely used - Y = yellowish
-C.I. 45380
-used as an acid (neg. charged)
-highlights + discriminates tissue elements
-binds to positively charged (cationic) tissue sites e.g. cytoplasmic proteins
-highly soluble in H2O
-water differentiates eosin