Dye chemistry and staining procedure

Introduction

The Hematoxylin and Eosin stain (H&E) is the most widely used histological stain because :
  • comparative simplicity
  • Ability to demonstrate clearly an enormous number of different tissue structures.
  • Hematoxylin stains cell nuclei blue black  shows good intranuclear detail.
  • Eosin stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange, and red.

Basics of Staining

  • Stains >chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue
  • Staining >treating tissues or cells with a series of reagents so that it acquires a color; no particles of dye are seen and the stained element is transparent

Purpose of Staining

  • Outlines tissues and cellular components.

  • Identification of tissues.
  • Establishes the presence or absence of disease processes.

Common Staining Method

 Most commonly used staining methods are –
  • Hematoxylin and Eosin staining in Histopathology
  • Gram’s Stain and Ziehl-Neelson staining in Microbiology.
  • Romanowsky staining in Hematology.
  • Papanicoloau staining in Cytology

Dyes:-

  • Essentially Aromatic benzene ring compounds or derivatives that possess the twin properties of color and ability to bind to tissue.

Mordant

  • A polyvalent metal ion which forms coordination complexes
  • with certain dyes.
  • A substance which acts as an intermediary between dye and tissue, thus increasing the affinity between them.
  • Strictly applicable to salts and hydroxides of divalent and trivalent metals.
  • The mordant dye complex ‘lake’ combines with tissue to form tissue-mordant-dye complex, which is insoluble.

Mordant - Dyes Application

  • Mordant is applied first, followed by the dye.
  • e.g Heidenhain’s iron hematoxylin
  • Mordant and dye are mixed together and then applied.
  • Commonly done in histotechnology
  • e.g Alum hematoxylin solutions
  •  Dye applied first, followed by the mordant. Hardly done in histotechnology.

Accentuators

Accentuators

  • Substances which increase the staining power of dye.
  • They increase the intensity & selectivity of stain.
  • e.g KOH in Lofflers methylene blue
  • phenol in carbol fuschin & carbol thionin.
Accelerators
  • Accentuators used in metallic impregnation technique for the nervous system.
  • e.g chloral hydrate
Trapping agents

  • Agents which holds dye combination with tissue or bacteria .
  • e.g tannic acid/iodine

Types of Staining Reaction

  • Absorption or direct staining – tissue penetrated by dye solution.
  • Indirect staining – using intermediate treatment with mordant
  • Physical staining – simple solubility of dye in element of tissue.
  • Chemical staining – formation of new substance e.g. PAS
  • Adsorption phenomenon – accumulation on the surface of the compound.

Types of Staining Method

Vital:- Applied to living tissue
Accomplished by injecting staining solution into some part of the body
Mixing of stain with living cells
Primarily used for research.

Routine:- Stains tissues with minimal differentiation
except between nucleus and cytoplasm.  Demonstrates general relationship among  cells, tissues and organs.
e.g Hematoxylin and Eosin stains

Special :- Demonstrates selective features of tissues
:
Particular cell products.
Microscopic intracellular and intercellular  structure.
e.g PAS stain for mucopolysaccharide.

Regressive
  • Tissue is initially overstained and then partially decolorized (differentiated) until the proper endpoint is reached.
  • Sharper degree of differentiation is obtained
  • The differentiation is controlled visually by microscopic examination.
  • Faster and more convenient .
Progressive
  • Tissue is stained for a predetermined time for adequate staining of the nuclei and leaves the background tissue relatively unstained.
  • Once the dye is taken up by the tissues, it is not removed.
  • The tissue is left in the dye solution until it retains the desired
  • amount of coloration.
  • The differentiation solely relies on the selective affinity of dyes for different tissue elements.

Differentiation


  • Removal or washing out of excess stain until the color is retained only by the tissue component that is to be studied.
  • Done with acid alcohol or ethyl alcohol
  • Exposure to air may oxidize and improve the process.

Requirements for Staining

  • All glassware should be thoroughly cleaned.
  • Correct solvent should be used.
  • Silver and osmic acid solutions should be kept in dark bottles.
  • Solutions like dilute ammonia should be freshly prepared.
  • Constituents of stain dissolved should follow the formula.
  • Alcoholic solutions of the stain should be kept in dark stoppered bottles.
  • All dyes should be filtered before use.

Eosin

Xanthine dyes which stains connective tissue and cytoplasm in varying intensity and shades (red to pink).

Available in the following types :
  • Eosin Y ( Eosin Yellowish, Eosin water soluble) – most widely available.
  • Ethyl Eosin (Eosin S, eosin alcohol soluble).
  • Eosin B ( Eosin Bluish, Erythrosine B).
Ethyl eosin and eosin B are now rarely used, although occasional old methods specify their use – e.g the Harris stain for Negri bodies.

The Hematoxylin and Eosin staining Technique  For Paraffin Sections


  • REMOVAL OF WAX.
  • HYDRATION WITH GRADED ALCOHOLS.
  • STAINING.
  • DIFFERENTIATION
  • BLUEING
  • COUNTERSTAIN WITH EOSIN
  • DEHYDRATION THROUGH GRADED ALCOHOL.
  • CLEARING IN XYLENE
  • MOUNTING UNDER A COVER SLIP.