Staining: Principles, Types & Methods

Staining
-Application of dyes on tissue sections to study the architectural patterns and physical
characteristics of cells

 Different tissues and cells have varying affinities for most dyes and stains.

AFFINITY:

 NUCLEUS-basic stain (basophilic)

 CYTOPLASM- acid stain(acidophilic)
GROUPS OF TISSUE STAINING:

1. HISTOLOGIC STAINING
Direct interaction with a dye or staining solution
Active tissue component is colored
Includes: micro-anatomical stains, bacterial stains, specific tissue stains
2. HISTOCHEMICAL STAINING
Study of tissue constituents through their chemical reactions
Perl’s Prussian blue reaction
Periodic Acid Schiff staining
3. IMMUNOHISTOCHEMICAL STAINING
Combination of immunologic and histochemical techniques
Detection of phenotypic markers that are detected by antibodies
Ex: monoclonal, polyclonal, fluorescent labeled or enzyme labeled antibodies

METHODS OF STAINING:

DIRECT STAINING
Uses aqueous or alcoholic dye solutions (ex. Methylene blue, eosin) to produce a color
INDIRECT STAINING
Uses a mordant or another agent to intensify the action of the dye used

MORDANT

 Serves as a link or bridge between the tissue and the dye

 Dye + mordant= insoluble tissue mordant dye complex

 Potassium alum, Iron

Staining: Principles, Types & Methods

ACCENTUATOR

 Not essential and does not participate to the chemical reaction of the tissue and dye

 Hastens the staining reaction by increasing the staining power and selectivity of dye

 KOH, phenol

PROGRESSIVE STAINING
-tissue elements are stained in definite sequence
-the staining with specific periods of time or until desired color is attained
-no decolorizer is applied
-the distinction of tissue detail relies solely on the selective affinity of the dye for various
cellular elements

REGRESSIVE STAINING
-Overstaining is done
-excess stain is removed or decolorized from unwanted parts of the tissue and until the
desired color is obtained

METACHROMATIC STAINING
-staining with a color that is different from that of the stain itself usually employed in
staining cartilage, connective tissue, epithelial mucins, amyloid and mast cell granules
Ex: Methyl violet, Bismarck brown, methylene blue, toluidine blue, cresyl blue

*H20 is important in metachromatic staining while -OH tends to lose the metachromatic stain

VITAL STAINS
 The selective staining of living cell constituents
 Demonstrates cytoplasmic structures (by engulfment of the dye particle, by staining of
pre-existing cellular components)

Nucleus is resistant to vital stains (staining of nucleus indicates dead)

2 types:

1. Intravital staining
-injected to any part of the body of animal
-common dyes: lithium, carmine and India ink
2. Supravital staining
-use immediately after removal of cell from the living body
-common dyes: Neutral red, Janus green, trypan blue, Nile blue,
thionine and toluidine blue
Staining: Principles, Types & Methods

H and E staining

I. HEMATOXYLIN
 Most commonly used for histologic studies
 Combined with mordants (such as alum and iron) to form the dye mordant tissue
complex
 Filter the stain prior to use
 Aluminum Hematoxylin
 Iron Hematoxylin

A. Aluminum Hematoxylin
 Routinely used in H and E staining
 Mordant used: alum
 Initially, it stains the nuclei reddish
 For progressive and regressive staining
 Examples:
Harris’ Hematoxylin
Ehrlich’s Hematoxylin
Delafield’s Hematoxylin
Mayer’s Hematoxylin
Cole’s Hematoxylin
Carazzi’s Hematoxylin

1. Harris hematoxylin
 routinely used in nuclear staining
 ripened with mercuric oxide
 cytology: nuclear stain in Pap’s smear
 staining of sex chromosomes
 addition of glacial acetic acid gives a precise nuclear staining
 Staining method: Regressive
2. Ehrlich’s Hematoxylin
 Excellent nuclear stain; stains mucins, recommended for bone and cartilage
 Not for frozen section
 Glycerin is added to slow oxidation and to prolong shelf life of hematoxylin
 Staining method: Regressive

3. Delafield’s hematoxylin
 Naturally ripened
 Similar longevity to Ehrlich’s

4. Mayer’s hematoxylin
 Chemically ripened with Sodium Iodate
 Primarily a regressive stain
Staining: Principles, Types & Methods

5. Cole’s hematoxylin
 Artificially ripened with alcoholic iodine

6. Carazzi’s hematoxylin
 Artificially ripened with potassium iodide
 For frozen sections

B. Iron hematoxylin
 Uses iron salts as both a mordant and a ripening agent
 Staining method: Regressive
 Diffentiator: acid alcohol
 Examples:
Weigert’s Hematoxylin
Heidenhain’s Hematoxylin
Loyez Hematoxylin

1. Weigert’s Hematoxylin
 Mordant/Oxidizer: Ferric Ammonium Chloride
 Standard Iron hematoxylin
 Used in demonstrating muscle fibers and Connective tissues

2. Heidenhain’s Hematoxylin
 Mordant/Oxidizer: Ammonium Chloride
 For nuclear and cytoplasmic inclusions; cytological stains
 Result: gray-black

3. Loyez Hematoxylin
 For frozen sections

C. Tungsten hematoxylin

 Mordant: 1% aqueous phosphotungstic acid

 Oxidizer: Potassium Permanganate
 Phosphotungstic Acid Hematoxylin

natural ripening achieved with light and air

Progressive stain
For CNS, general tissue

D. Lead hematoxylin

E. Copper hematoxylin
Staining: Principles, Types & Methods

II. Eosin

 A red acid (xanthene) dye

 Routinely used as a counterstain after hematoxylin and before methylene blue
 Stains connective tissues and cytoplasm differentially
 It is commonly used as a background stain

3 forms:
Yellow (Eosin Y)
Eosin B, Erythrocin B
Eosin S, Ethyl eosin

Factors that affect H and E stains:

 Concentration of the Dye
 Temperature
 pH of the Staining Solution: can have a direct impact on the ability of a dye to bind with its
intended tissue element.
 Tissue fixation: Unfixed tissue elements have limited binding sites for dyes

Other stains:

METHYLENE BLUE- used as a bacterial stain for evaluation and grading of milk, for
diagnosis of diphtheria, for vital staining of nervous tissue, and as an indicator of
differentiation of organisms in bacteriology

METHYLENE VIOLET- metachromatic dye formed when Methylene blue is heated in alkali
carbonate, coloring nuclei of WBCs reddish purple in the presence of methylene blue

TOLUIDINE BLUE- nuclear stain recommended for staining Nissl granules or

chromatophilic bodies

CRYSTAL VIOLET- nuclear stain used for staining amyloid in frozen sections and platelets in
blood

ANILINE BLUE- cytoplasmic stain for counterstaining of epithelial sections

BASIC FUCHSIN- plasma stain used for deep staining of acid-fast organisms and for
mitochondria

GIEMSA STAIN- used for staining blood to differentiate WBCs

CELESTINE BLUE- used for routine staining of fixed sections

Staining: Principles, Types & Methods

MALACHITE GREEN- used as a contrast stain for Ascaris eggs and RBCs, and as a bacterial
spore stain; also used as a decolorizer and counterstain

METHYL GREEN- stains chromatin green in the presence of an acid

BISMARCK BROWN- used as a contrast stain in Gram’s, AFS, and Papanicolau methods; for
staining diphtheria organisms

PRUSSIAN BLUE- used in intravital staining

ORCEIN- excellent stain for elastic fibers, recommended for dermatological studies

PICRIC ACID- used as a contrast stain to acid fuchsin for demonstration of CT, as a
cytoplasmic stain, counterstain to crystal violet, as fixative and decalcifying agent

CARMINE- used as a chromatin stain in smear preparation

 Best’s Carmine Solution- with AlCl to stain glycogen
 Mucicarmine- to stain mucin
 Mayer’s Carmalum Solution- mordanted basic dye staining acidic substances

IODINE- stains amyloid, cellulose, starch, carotene and glycogen

ALCIAN BLUE- stains acid mucopolysaccharides

NEUTRAL RED- basic dye used for observing cell granules and vacuoles of phagocytic cells

CONGO RED- used as 4% aq. Solution in Krajian’s method of staining elastic tissues,
amyloid and myelin

Janus Green B- used for staining mitochondria during intravital staining

VICTORIA BLUE- used for staining neuroglia in frozen sections

NIGHT BLUE- used as a substitute for carbol-fuchsin in AFS

ACRIDINE RED 3B- used to demonstrate deposits of calcium slats and possible sites of
phosphatase activities

ACRIDINE ORANGE- permits discrimination between dead and living cells, giving green
fluorescence for DNA and red for RNA

BENZIDINE- used for staining Hemoglobin

RHODAMINE- with osmic acid, fixes and stains blood and glandular tissues
Staining: Principles, Types & Methods

GOLD SUBLIMATE SOLUTION- made up of gold chloride and HgCl2; used for metallic
impregnation

OSMIUM TETROXIDE- used as a fixative; demonstration of fats(black)

AgNO3- used in 10% solution for demonstration of spirochetes, Reticulum and other
fibers

RESTAINING OF OLD SECTIONS

 Old bleached or faded sections may be restained

 Slide immersed in xylene for 24 hours or gently heated until mounting medium begins to
bubble, coverslip is removed by lifting with a dissecting needle
 Section is placed in xylene for 30 minutes to remove the remaining balsam, then to water
 Placed in 0.5 % KMnO4 solution for 5-10 minutes, rinsed in tap water, immersed in 5%
oxalic acid for 5 minutes until section is decolorized, rinsed in tap water for 5 minutes and
restained.