STAINING PART 1

Purpose of staining

• Enhancing contrast and visualization of cellular components

• Studying architectural pattern of tissue and physical characteristics of cell

• Establishing presence & absence of disease

Factors influencing DYE BINDING

• pH of soln

• inc. temp

• inc. concentration of dye molecules

• presence of other salts

• types of fixatives

HISTOLOGICAL STAINING

tissue constituents are demonstrated in sections by DIRECT INTERACTIONS WITH DYE OR STAINING SOLN

HISTOCHEMICAL STAINING

tissue constituents are studied thru CHEMICAL REACTIONS that will permit microscopic localization of specific tissue substances

IHC STAINING

combination of immunologic and histochemical techniques to detect phenotypic markers under microscope

BASIC DYES

• “cationic dyes” ++++

• stains acidic portion

ACID DYES

• “anionic dyes” - - - -

• stains basic portion

METHODS OF STAINING

• Direct

• Indirect

• Progressive

• Regressive

• DIRECT STAINING

• uses aqueous or alcoholuc dye solutions

• ONLY ONE DYE, which is washed away after 30-20 secs

INDIRECT STAINING

action of dye is intensified by adding another agent or mordant

INTEGRAL part of staining reaction

MORDANT

MORDANT

bridge between dye and tissue

ACCENTUATOR

• NOT ESSENTIAL to the chemical union of the tissue and dye

• merely ACCELERATES the reaction

PROGRESSIVE STAINING

• tissue elements are stained in a DEFINITE SEQUENCE, and the staining soln is applied for a specific period of time

• NOT WASHED OR DECOLORIZED

REGRESSIVE STAINING

• tissue is first overstained to obliterate cellular details

• excess stain is REMOVED OR DECOLORIZED

under regressive staining and is the most common method utilized for microanatomical studies of tissues

Hematoxylin & Eosin

SELECTIVE removal of excess stain = specific substances are stained distinctly from the surrounding tissue

DIFFERENTIATION/DECOLORIZATION

uses more than one chemical stain to differentiate between various microorganisms or structures/cellular components of a single organism

Ex. Gram Staining

DIFFERENTIAL STAINING

(under differential staining)

acts as a differentiator for both BASIC and ACIDIC DYES

ALCOHOL

(under differential staining)

acts as a differentiating agent

MORDANT

staining with a color different from that of the stain itself (metachromasia)

METACHROMATIC STAINING

dye stains tissues or cells similar in its own true color

ORTHOCHROMATIC STAINING

process where specific tissue elements are demonstrated by colorless solution of metallic salts which are thereby reduced by the tissue, producing OPAQUE, BLACK DEPOSITS on surface of tissue or bacteria

Ex. Ammoniacal Silver reduced by argentaffin cells = BLACK DEPOSITS

METALLIC IMPREGNATION

process where dye can penetrate/stain the living cells w/o killing them

VITAL STAINING

TYPES OF VITAL STAINING

• Intravital Staining

• Supravital Staining

done by INJECTING the dye into any part of the animal body, producing specific coloration of certain cells

INTRAVITAL STAINING

dye is applied IN VITRO to the living cells

SUPRAVITAL STAINING

Common dyes for INTRAVITAL STAINING

1. Lithium

2. Carmine

3. India Ink

si Lithium nag Car pa India

commin dyes used in SUPRAVITAL STAINING

1. Neutral red

2. Janus green

3. Trypan blue

4. Nile blue

5. Thionine

6. Toluidine blue

si Janus nag thio sa kalipay kay giregalohan syag neutral red ug 3 ka blue tingz

BEST vital dye

Neutral red

recommended for mitochondria

Janus green

NATURAL DYES (plants and animals)

• Hematoxylin

• Cochineal dyes

• Orcein

• Saffron

most valuable staining agent and most widely used as NUCLEAR STAIN (basic dye)

Hematoxylin

Hematoxylin is extracted from the core wood of a Mexican tree called

Haematoxylin campechianum

activr coloring agent (hematoxylin) and is formed by ripening

Hematin

types of oxidation/ripening

• Natural

• Artificial

exposes substance to AIR and SUNLIGHT, oxidizing hematoxylin

takes about 3-4 months

NATURAL OXIDATION

uses oxidizing agents to accelerate process (oxidation)

ARTIFICIAL RIPENING

OXIDIZING AGENTS

1. H202

2. Mercuric oxide

3. Potassium permanganate

4. Sodium iodate

removing excess hydrogen ion from stain

soluble hemalum is converted to insoluble form

nucle: CRISP BLUE

BLUING

• HEMATOXYLIN STAINS

• Aluminum/Alum

• Iron

• Tungsten

• Molybdenum

• Lead

• Copper

progressive and regressive staining

usually counterstained with eosin, congo red and safranin

Mordant: Aluminum (Aluminum K sulfate or Aluminum ammonium sulfate)

Aluminum (Alum) Hematoxylin

Types of Aluminum Hematoxylin

• Ehrlich

• Harris

• Delafield’s

• Cole’s

• Mayer’s

• Gill

EH D CuMaG

naturally ripened for about 2 months

NOT IDEAL for FROZEN sections

Ehrlich Hematoxylin

Ehrlich Hematoxylin

(blank) may be added to hasten process

(blank) is added to act as an oxidation stabilizer - prolongs shelf life

• sodium iodate

• glycerin

In Ehrlich’s Hematoxylin, mucopolysaccharidesubstances are stained:

intensely blue

widely used for routine nuclear staining in exfoliative cytology and sex chromosomes

is stable and can be stored up to 6 mos

Harris Hematoxylin

In Harris Hematoxylin, originak formula uses mercuric oxide as oxidizer but is replaced by:

sodium iodate

Natural ripening (4 to 6 months) - longer ripening

Delafield’s Hematoxylin

Artificially ripened with alcoholic iodine

used after celestine blue

Cole’s Hematoxylin

Chemically ripened with sodium iodate

Mayer’s Hematoxylin

ripened with potassium iodate

Gill Hematoxylin

stains mucin

sensitive to acid: may stain gel adhesives and glass slide

Gill

used in gill, to prevent formation if surface precipitate

ethylene glycol (ethanediol)

types of iron hematoxylin

• Weigert’s

• Heidenhain’s

• Loyez

• Verhoeff

standard iron hematoxylin; used in demonstrating muscle fibers and connective tissues

(Ferric chloride)

Weigert’s

stains tissue jet black

Heidenhain’s

used for demonstration of both nuclear and cytoplasmic inclusions

(Ferric Ammoniun Sulfate)

Heidenhain’s