Special Stains

Ju n a l l y Je rca M a e La y s o n , RM T , M PH
Special Stain
Is a term used to refer to many alternative staining techniques
aside from the traditional H&E

Is a process that generally employ a dye or chemical that has an

affinity for the particular tissue component that is to be
demonstrated

Allows the presence/or absence of certain cell types, structures

and/or microorganisms to be viewed microscopically
Special Stain
Purpose and indications:
To visualize selected tissue elements, entities and
microorganisms
Provides valuable information in the evaluation of
numerous abnormal or disease conditions
Satisfactorily demonstrate tissue components or
organisms for which they were designed
Connective Tissue Stains
Collagen
Elastic Fibers
Reticulum
 von Gieson’s Stain
Simplest method of differential staining
of collagen and other connective tissue
Connective Uses a mixture of picric acid and acid
Tissue Stains fuchsin
Collagen Fibers ▶ Collagen – pink or deep red

▶ Nuclei – brownish black to black

▶ Muscle, cytoplasm, rbc and fibrin –

yellow

Fixatives: Mercuric chloride or formol saline

 Masson’s Trichrome stain
Often used to stain connective tissue
Trichrome - produces three colors
Connective
▶ Nuclei and other basophilic
Tissue Stains structures – blue
Collagen Fibers ▶ Cytoplasm, muscle, rbc and keratin –
bright-red
▶ Collagen – green or blue
 Masson’s Trichrome stain
Consists of sequential staining:
1. Iron hematoxylin (Weigert’s or
Connective Heidenhain’s): nuclei (black)
Tissue Stains 2. Biebrich scarlet: cytoplasm & muscle
(red)
Collagen Fibers 3. Aniline blue/Aniline light green:
collagen (blue or green)

Fixatives: Bouin’s, Zenker’s, Formol-mercury,

Zinc formalin, Picro-mercuric alcohol
 Gomori’s One Step Trichrome
Stain
One step method where a plasma stain
Connective


(chromotrope 2R) and a connective tissue fiber

Tissue Stains stain (fast green FCF, light green or aniline blue)
are combined in a solution of phosphotungstic
Collagen Fibers acid to which glacial acetic acid has been added
 Muscle fibers – red

 Collagen – green

 Nuclei – blue to green

Fixatives: Bouin’s solution

 Russell’s modification of the
Movat pentachrome stain
Connective  Alcian blue – acidic mucosubstances
Tissue Stains  Iron hematoxylin – elastic fibers

Collagen Fibers  Crocein scarlet – stain muscle,

cytoplasm, collagen and ground
substance
 Acid fuchsin

 Alcoholic safran

Fixative: 10% neutral buffered formalin

 Russell’s modification of the
Movat pentachrome stain
Connective
Tissue Stains  Nuclei and elastic fibers – black
 Collagen – yellow
Collagen Fibers  Ground substance and mucin – blue

 Fibrinoid, fibrin – intense red

 Muscle - red
 Mallory’s Aniline Blue stain
 Often used to differentiate acidophilic
extracellular fibers from the acidophilic
Connective cytoplasm
Tissue Stains  Is NOT absolutely differential because it also
stains hyaline fibrils, fibroglia fibrils, smooth
and striated muscle fibers and amyloid
Collagen Fibers  Acid fuchsin

 Aniline blue

 Orange G

 Phosphotungstic acid

Fixatives: Zenker’s
 Mallory’s Aniline Blue stain
 Nuclei, fibrin, muscle fibers – red
 Collagen – blue
Connective
 Cartilage, bone, mucus – varying shades
Tissue Stains of blue
Collagen Fibers  Blood, myelin – yellow

 Elastic fibers – pale pink/yellow or

unstained
 Azocarmine Stain
 Heidenhain’s modification of Mallory’s
aniline blue stain
Connective  Azocarmine dye – counterstain
Tissue Stains  Shows minute details of connective

Collagen Fibers tissue and of renal glomerular basement

membrane
 Amyloid connective tissues and mucus colloid
– deep blue
 Nuclei - red
 Verhoeff’s Elastic Method
 Most commonly used: quick and produces
intense staining of elastic fibers that are not
easily differentiated by H&E staining
Connective 1. Overstaining with a compound solution:
Hematoxylin
Tissue Stains
◦

◦ Ferric chloride – mordant and oxidizing agent

◦ Iodine – mordant and oxidizing agent
Elastic Fibers ◦ Elastin remains stained, other tissue elements are
decolorized

2. Differentiation
◦ Ferric chloride – break down tissue-mordant complex
◦ Sodium thiosulfate – remove excess iodine
◦ Van Gieson – produce contrast with hematoxylin stain

 Fixative: formalin
 Verhoeff’s Elastic Method

 Elastic fibers – black

Connective
 Nuclei – gray to black
Tissue Stains
 Collagen – red
Elastic Fibers  Cytoplasm and muscle - yellow
 Weigert’s Resorcin-Fuchsin Elastic
Tissue Stain
 Weigert’s stain: basic fuchsin, resorcin
Connective and ferric chloride
Tissue Stains  Counterstain: neutral red, H&E or

Elastic Fibers hematoxylin and van Gieson’s stain

 Elastic fibers – brown to purple or blue-black
with methyl violet on a clear background

 Fixative: formalin or alcohol

 Van Gieson stain
 Regressive method: sections are
overstained (hematoxylin ferric chloride –
iodine) and then differentiated (ferric
Connective chloride or iodine)
Tissue Stains  Sodium thiosulfate – removes excess iodine
Elastic Fibers Verhoeff-van(VVG) staining method
 Method is used to identify elastic fibers in
tissues such as skin, aorta on formalin-fixed,
paraffin-embedded tissues or in frozen
sections
 Elastic fibers – blue-black
 Background – yellow
 Aldehyde fuchsin elastic stain
 HCl and fresh paraldehyde are added to
an alcoholic solution of basic fuchsin
Connective  Elastic fibers – deep blue to purple
Other tissue elements - green
Tissue Stains 

Elastic Fibers Luna Staining Method and

Protocol on Elastic Fibers and Mast
Cells
 Used to identify elastic fibers and mast
cells on formalin-fixed, paraffin-
embedded tissue sections, and frozen
sections
 Orcein (Taenzer-Unna Orcein
Method)
 Orcein – naturally occurring vegetable dye
used to stain elastic fibers
Connective Can demonstrate the finest and the most
Tissue Stains

delicate fibers found in the skin
Elastic Fibers  Counterstain: methylene blue or alum
hematoxylin

Krajian’s Technique
 Rapid method to stain elastic fibers, fibrin
and amyloid
 Congo red is used
 Bielschowskys Method
a silver stain to demonstrate
neurofibrillary tangles, nerve fibers and
Connective senile plaques in Alzheimer's disease
Tissue Stains  axons, plaque neurites and tangles - black
 Background - yellow to brown
Reticulum  plaque and vascular amyloid - generally
(Reticular brown to dark brown

Connective
Fixative: 10% Neutral Buffered Formalin
Tissue)
 Wilder’s Stain
A modification of Bielschowsky stain
Reticulin fibers – black
Connective
Nuclei – red
Tissue Stains
Background – grey
Reticulum
(Reticular Fixative: 10% Neutral Buffered Formalin
Connective
Tissue)
 Gomori’s Silver Impreganation
Stain for Reticulin
Optimal pH for maximum uptake of
Connective silver ions is pH 9.0
Tissue Stains Two most common reticulin stains:
Reticulum • Gomori’s stain
• Gordon & Sweet stain
(Reticular
Connective  Reticulin fibers - black

Tissue)
Stains for cytoplasmic granules
Argentaffin and Melanin pigments
Mast cell granules
 Fontana Masson
 Argentaffin – possess the ability to bind silver
Stains for from a solution and to reduce it to visible
metallic silver without the need for a separate
cytoplasmic reducing agent
granules  Melanin – ability to reduce solutions of
ammoniacal silver nitrate to metallic silver
Argentaffin and
Melanin Primary stain: silver solution
Counterstain:1% neutral red
pigments ▶ Melanin – black

▶ Argentaffin granules – black

▶ Nuclei - red

Fixatives: 10% neutral buffered formalin

 Schmorl’s method
Sections are immersed in a solution
Stains for containing ferric chloride and potassium
cytoplasmic ferricyanide
granules Expected product = ferrous ferricyanide
Argentaffin and = Turnbull’s blue (blue pigment)
Melanin
▶ Reaction is NOT melanin-specific, may
pigments stain argentaffin, chromaffin cells and
some types of lipofuchsin
 Dominici’s Method
used for the demonstration of
Stains for metachromatic granules of mast
cytoplasmic
granules cells
Acid fuchsin – orange G solution
Mast cell
Toluidine blue solution
granules Granules of mast cells – blue
Other tissue elements – pink to rose

Fixative: Zenker’s
 Mallory’s Phloxine Methylene
Stains for
Blue
cytoplasmic Phloxine is employed in place of
granules eosin because it gives a more
Mast cell brilliant color
Also used to demonstrate
granules connective tissue
Stains for Fats
 Sudanophilia – property of tissues to be stained with fat or
oil-soluble dyes
 Oil Red O
 Stains neutral fats and lipofuchsin
well
Stains for  Preferred choice of stain because of
fats its intensity of coloration
 Lipid – red
 Nuclei – blue

Fixative: fresh frozen or frozen

sections post-fixed in NBF
 Sudan Dyes
 Group of lipid soluble solvent dyes often
called lysochromes
Stains for  Sudan III – first dye introduced in 1896
fats - Predominantly used to stain triglycerides in
animal tissues

 Sudan IV – Scharlach R in 1901

 Most commonly used stain, producing a rapid
and permanent coloration of lipid
 Sudan Dyes
 Sudan Black B – introduced in 1935
 Most sensitive and versatile of all Sudan dyes
Stains for  Stains phospholipids and neutral fats
fats  Does NOT stain crystalline cholesterol

◦ Lipids – blue black; red for Sudan IV

◦ Nuclei – red; blue/black for Sudan IV

Fixative: Formalin
 Osmic Acid stain for Fat
 NOT a dye but is an unstable oxide which is
reduced to a permanent black substance by
unsaturated fats and fatty acids
Stains for
fats Nile Blue Sulfate Method for Fats
 Dye capable of differentiating two lipid classes
simultaneously
 Used as a preliminary indicator of the type of
lipid present in the tissue section
 Present as a minor component of non-
fluorescent lipid stain Nile blue
 Red oxazine – dissolves neutral lipids
 Blue oxazine – basic and reacts with phospholipids and
free fatty acids
Stains for carbohydrates &
connective tissue ground substance
Glycogen
Acid mucopolysaccharides
Connective tissue mucin & Cryptococcus
Amyloid
 Periodic Acid-Schiff Reaction
 Schiff’s reagent – primary stain
 Counterstain (optional) – Harris hematoxylin
with acetic acid
Stains for  PAS-positive substances – red or magenta red
carbohydrates  Nuclei – blue
Glycogen  Mucoproteins – most common PAS positive substances
 Mucin and intestinal mucoid secretions,
tracheobronchial aspirates and hyaline casts (kidney)
 Is a useful indicator for glycogen when the
technique incorporates a diastase digestion
stage

 Fixative: 10% NBF or Bouin’s solution

 Best Carmine Stain
 Is a good staining technique due to the affinity
of alkaline carminic acid for glycogen,
Stains for producing a bright red color
carbohydrates  Counterstain: Ehrlich’s hematoxylin, colors the
nuclei blue
Glycogen  NOT highly specific for glycogen
 Nuclei – blue or grayish blue
 Glycogen – pink to red
 Mucin – weak red
 Acid mucopolysaccharides
 Are polysaccharides with hexuronic acid as
secondary carbohydrate constituent, bound to
sulfuric acid esters and proteins
Stains for  Comprise mainly of hyaluronic acid, heparin
carbohydrates sulfate and chondroitin sulfate
 Comprise the intercellular of ground substance
e.g. reticulum, elastin and collagen
 Especially found in synovial fluid, vitreous
humor and Wharton’s jelly (umbilical cord)

 ONLY group of carbohydrate compounds that

are not strongly PAS positive
 Metachromatic staining
a. Toluidine blue and Azure A
b. Uranyl nitrate – Azure method

Stains for ◦ Azure A - most useful metachromatic dye for acid mucin
carbohydrates ◦ Toluidine blue - basic thiazine metachromatic dye with
high affinity for acidic tissue components and nucleic
Acid acids
mucopolysaccharides ◦ Mucopolysaccharide – red purple
◦ Tissue background - blue

Alcian blue technique

 Histological dye that forms electrostatic bonds with
certain tissue components containing either carboxyl or
sulfate groups
 Colloidal iron technique

Aldehyde fuchsin stain

Stains for ◦ First introduced by Gomori in 1950 as an elastic
carbohydrates tissue stain
Acid ◦ Employs basic fuchsin plus aldehyde to
demonstrate sulfur-containing compounds
mucopolysaccharides
Mucicarmine stain
◦ Aluminum hydroxide plus carmine
◦ Southgate’s mucicarmine technique – useful for
staining encapsulated fungi e.g. Cryptococcus
neoformans
 Fluorescent acridine orange
technique
 Acridine orange - fluorescent stain
Stains for (fluorochrome) that can be used to
carbohydrates demonstrate acid mucins
 Disadvantage: temporary and will last only
Acid for about two hours once the section is
mucopolysaccharides mounted
 Mayers mucicarmine stain
empirical stain
stains the spores and hyphae as pale to dark
Stains for red
carbohydrates used to demonstrate capsule of
Cryptococcus neoformans and other fungi
connective
tissue mucin & ◦ Nuclei – blue
◦ Mucins – red
Cryptococcus ◦ Background – pink

Southgate’s mucicarmine technique – useful

for staining encapsulated fungi e.g.
Cryptococcus neoformans
 Liebs Method –crystal violet
◦ Amyloid - Purplish Violet
◦ Other tissue elements – Blue
Stains for
carbohydrates Fixative: 10% BNF
Amyloid
Silver reduction method
Stains for pigments & minerals
Iron pigments
Calcium
Bile pigments
 Gomori’s Prussian Blue stain
 used to detect loosely bound ferric iron
Stains for in tissue sections, bone marrow smears
pigments & and blood smears
minerals histochemical reaction is sensitive
Iron pigments enough to demonstrate even minute
amounts of iron deposits in blood cells,
bone marrow, spleen and liver
 Iron pigments – bright blue
 Nuclei – red
 Cytoplasm – pink to rose
 Von Kossa’s Method
 used to detect the presence of abnormal
Stains for calcium deposits in the body
pigments &  principle is based on the transformation of
minerals calcium salts into silver salts: calcium ions,
bound to phosphates, are replaced by silver
Calcium ions brought by a solution of silver nitrate

 Calcium salts - black

 Nuclei - red
 Cytoplasm - pink
 Steins stain
Combination of iodine, sodium
Stains for thiosulfate and nuclear fast red
pigments &
minerals
Bile pigments
Nervous tissue stains
 Glial fibers –Holzers stain
Nervous Glial cells that have been stained with
tissue crystal violet are resistant to
stains decolorization due to the alkalinity of
the aniline-chloroform mixture.

Glia fibers - deep violet-blue

Background - pale violet

Fixative: 10% formalin

 Nerve fibers/endings –
Bodian’s Method
Nervous  Protargol-S (silver proteinate) is used with the
tissue addition of copper metal
stains  Copper replaces the silver in the connective
tissue then silver is reduced with hydroquinone
to the visible metallic form

Nerve fibers - black

 Myelin Sheath –Weil –Weigert’s
Stain
Nervous  modification for paraffin sections of the
tissue Weigert-Pal-Kulschitsky technique
stains  involves the reduction of chrome salt to
chromium dioxide by myelin

◦ Myelin-containing structures – black

◦ Red blood cells – black
◦ Nuclei – blue
◦ Background - clear or yellow
Stains for bacteria, fungi and
organisms
Acid fast microorganisms
Gram positive /gram negative bacteria
Fungi
 Kinyoun’s stain
Stains for Unlike the Ziehl-Neelsen modified acid-
bacteria, fast stain, the modified Kinyoun acid-fast
fungi and stain does not require heating the
organisms reagents used for staining
Acid fast
microorganisms Primary stain: Kinyoun carbolfuchsin
Counterstain: methylene blue

Mycobacteria – red
Background - blue
 Fite Faraco stain
Stains for  Modification of Ziehl-Neelsen method which
bacteria, utilizes a weaker acid for Mycobacterium leprae
fungi and
organisms  Specific way to identify mycobacteria based on
the ability of the lipoid capsule of acid-fast
Acid fast organism to take up carbolfuchsin and resist
microorganisms decolorization with dilute mineral acid

 M. leprae and other acid fast bacteria – bright

red
 Background – light blue
 Brown & Brenn Technique
Stains for  Used to demonstrate gram-negative and gram-
bacteria, positive bacteria in tissue
fungi and  Cell wall consisting of peptidoglycan layer will
organisms take up the crystal violet
 Gram-negative bacteria to take up the basic
Gram positive fuchsin stain due to a thin peptidoglycan wall
/gram negative ◦ Gram-positive bacteria - blue
◦ Gram-negative bacteria - red
bacteria ◦ Nuclei - red
◦ Background - yellow
 McCallum Goodpasture
Stains for Technique
bacteria,
fungi and
organisms Primary stain: Goodpasture’s
Gram positive solution
/gram negative Counterstain: Crystal Violet
bacteria
a stain for bacteria using aniline
fuchsin
 Gomori’s methenamine silver
Stains for nitrate
bacteria,  Used to identify fungi
fungi and  The mucopolysaccharide components of the
organisms fungal cell wall are oxidized to release aldehyde
groups. The aldehyde groups then react with
Fungi the silver nitrate, reducing it to a metallic silver,
rendering them visible.

 Fungi - black
 Background - green
 Gram’s stain
Stains for  Can be used to demonstrate pathogenic fungi
bacteria, in tissue sections
fungi and
organisms  Primary stain: Crystal violet
 Counterstain: Safranin
Fungi
 yeast organisms will stain dark blue/purple,
and budding will be easy to detect
 Toluidine blue
Stains for -for Helicobacter: dark blue against a variably
bacteria, blue background
fungi and
organisms Dieterle method
-for Spirochetes and Legionella: brown to black
Others against a pale yellow or tan background

Warthin-Starry Method
-for spirochetes: black against a golden yellow
background
 Giemsa
Stains for -for blood and marrow parasites (Leishmania,
bacteria, Malaria and Trypanosomes)
fungi and
organisms Mann’s or Sellers stain
-permit differentiation of rabies inclusions from
Others other intracellular inclusions
-with these stains, Negri bodies appear magenta
in color