NATURAL DYES

1. Hematoxylin – Mexican heartwood tree – “Hematoxylin campechianum”

a. Alum Hematoxylin – PROGRESSIVE
i. Ehrlich’s – Sodium Iodate
ii. Harris’ – Mercuric Chloride
iii. Cole’s – Alcoholic Iodine
iv. Mayer’s – Sodium Iodate – can be used for REGRESSIVE staining
b. Iron Hematoxylin – REGRESSIVE (acid-alcohol as differentiating agent)
i. Weigert’s – Ferric Chloride
ii. Heidenhain’s – Ferric Ammonium Sulfate
iii. Phosphotungstic Acid
c. Copper Hematoxylin – for spermatogenesis

Results:
• RBCs – orange
• Collagen and cytoplasm – pink
• Nucleus and acidic structures – blue

2. Cochineal dyes – Female cochineal bug – “Coccus cacti”

• Treated with alum – carmine
o With picric acid – picocarmine
o With aluminum chloride – Best’s carmine
• For glycogen

3. Orcein – Vegetable dye from lichens

• For elastic fibers
• Add lime and soda – LITMUS

4. Saffron

SYNTHETIC DYES – “Coal Tar Dyes”

• Derived from benzene – “Aniline dyes”
• Three groups:
o Acid dyes
§ Picric acid
§ Fixative: trichloroacetic acid, picric acid, chromium-fixed
§ Stains basic cell structures
o Basic dyes
§ Methylene blue
§ Fixative: mercuric chloride, formalin
§ Stains acidic cell structures
o Neutral dyes
§ Romanowsky dyes, Giemsa, Irishman’s stain
§ Fixative: ethanol, acetic acid
§ Stains both acidic and basic cell structures

EOSIN
1. Eosin Y – yellowish
2. Eosin B – bluish or imperial red

ROMANOWSKY STAINS
• Wright’s, Jenner’s, Leishman, Giemsa
• Can detect blood-borne parasites
• Can differentiate types of WBCs

OTHER STAINS
• Acid Fuchsin-Picric Acid – “Van Gieson’s Stain” – Connective Tissue
• Acid Fuchsin – Masson Stain – Collagen, Smooth Muscle, Mitochondria
• Acridine Orange – DNA – Green; RNA – Red
• Acridine Red 3B – Calcium salts
• Alcian Blue – Acid mucopolysaccharides
• Alizatin Red S – Calcium salts
• Aniline Blue – Epithelial cells (Counterstain)
• Azocarmine – Nuclei – DEEP red; cytoplasm – PALE red
• Basic Fuchsin – Acid-fast organisms, mitochondria, smooth muscles
o Feulgen’s and Schiff’s – Aldehydes
o Van Gieson’s – Connective tissues, mucin, elastic tissues
• Benzidine – Hemoglobin

• Bismarck Brown – Diphtheria organisms

• Carmine – Aluminum
o Best Carmine – Glycogen
• Celestine Blue – Alternative to Iron Hematoxylin
• Congo Red – Amyloid
• Cresyl Violet – Nervous tissues (Nissl Bodies)

• Crystal Violet – Amyloid in frozen, platelets

o Gentian Violet (Crystal violet, methyl violets, and dextrin)
• Ethidium Bromide – DNA (red-orange), marker of apoptosis
• Giemsa Stain – Leukocytes, blood-borne parasites
o Consists of methylene blue and eosin
• Gold sublimate – Metallic impregnation
• Iodine – “oldest of all stains” – Amyloid, cellulose, starch, carotenes, glycogen

• Gram’s iodine – Bacteria differentiation

• Lugol’s Iodine – Starches
• Janus Green B – Mitochondria (Intravital stain)
• Malachite Green – Ascaris eggs, RBCs, bacterial spore stain
• Masson’s Trichrome – RED – keratin, muscle fibers
– BLUE or GREEN – collagen and bone
– PINK – cytoplasm
– BLACK – cell nuclei

• Methyl Green – Chromatin (green)

• Methylene Blue – Plasma cells, vital stain for nervous tissue
o Polychroming – oxidation process – BLUE – Nuclei
– REDDISH-VIOLET – connective tissue, mucin
• Mallory’s Phloxine Methylene Blue Stain – Originally: EMB
• Methylene Violet – Leukocytes (reddish-purple)
o Heated methylene blue
• Nile red – Lipids (red)
o Boiled Nile blue with sulfuric acid

• Oil Red O – Neutral lipids and fatty acids

• Orcein – Elastic fibers
• Osmium Tetroxide – Unsaturated lipids and myelin (black)
• Periodic Acid Schiff (PAS) – Glycogen, mucin, mucoprotein, glycoprotein, basement
membranes, capsules, blood vessels, fungi
• Phosphotungstic Acid – Viruses, nerves, polysaccharides, striated muscle fibers

• Picric Acid – Connective tissue (Van Gieson’s Stain)

• Prussian Blue – Blood and lymph vessels (intravital staining)
• Rhodamine B – Blood and glandular tissues
• Safranin O – RED – nuclei; YELLOW – collagen
• Silver Nitrate – Spirochetes, reticulum

• Toluidine Blue – Nissl granules, chromophilic granules, mast cells

• Van Gieson Stain – Collagen – PINK; elastic fibers – BLACK
• Victoria Blue – Neuroglia (frozen section)
• Von Kossa Stain – Phosphates and carbonates, calcium
• Wright Stain – Basophilia, azurophilia, acidophilia, neutrophilia
OIL SOLUBLE DYES (LYSOCHROMES)
• Sudan Black B – MOST SENSITIVE - Black
• Sudan IV (Scharlach R) – Red
• Sudan III – Lighter Orange

CHIEF SOLVENTS USED FOR STAINS

1. Distilled water
2. Alcohol
3. Aniline water
4. Phenol

STAINING OF CARBOHYDRATES STAINING OF PROTEINS AND NUCLEIC ACIDS

1. Periodic Acid Schiff – oxidation of carbohydrates 1. Alkaline Fast-Green
• Polysaccharides • Basic Proteins (Protamines and
• Neutral mucus substances histones)
• Tissue basement membranes 2. Peracetic Acid-Alcian Blue
• Fungal organisms • Cystine
• Helps in the diagnosis of: • Cysteine
o Glycogen storage diseases 3. Alcian Blue-PAS
o Tumors • Proteoglycans
o Fungal infections
o Basement membranes STAINING OF NUCLEIC ACIDS
1. Feulgen Staining
2. Schiff Reagent – Essential: Basic fuchsin • DNA – red purple
(rosanilin, pararosanilin, magenta II) 2. Methyl Green-Pyronin
• DNA – green
STAINING OF GLYCOGEN • RNA – rose-red
1. PAS – Red 3. Fluorescein – blue
2. PAS with Diastase Method – Red 4. Rhodamine – orange-red
3. Langhan’s Iodine Stain – Brown 5. Acridine Orange
4. Best carmine method – Red • DNA – yellow-green
• RNA – orange-red
STAINING OF MUCIN 6. Acriflavine
1. Acid mucopolysaccharide • DNA – yellow
• Toluidine Blue – Red-purple
• Alcian Blue – Blue STAINING OF ENZYMES
• Alcian Blue-PAS Hematoxylin – Blue 1. Alkaline phosphatase
• Gomori’s Aldehyde Fuchsin • Gomori Calcium – Brownish-black
• Mucicarmine Stain – red 2. Acid phosphatase
o Also for C. neoformans • Gomori Lead – Black
• Colloidal Iron Technique 3. 5’-nucleotidase
• Fluorescent Acridine Orange – Black • Lead – Blackish-brown
4. Nonspecific esterase
2. Neutral mucopolysaccharides • Alpha-napthyl acetate – Reddish-brown
• Alcian Blue-PAS Hematoxylin – Magenta • Indoxyl acetate – Blue
5. Specific esterase
STAINING OF LIPIDS • Chloroacetate esterase – Reddish-brown
1. Sudan Black B – blue black 6. Acetylcholinesterase – Red/brown
2. Sudan Red VII B 7. Monoamine oxidase
3. Sudan III • Tetrazolium – Bluish-black
4. Sudan IV (Scharlach B) – red 8. Phosphorylase – Blue/Black
5. Oil Red O – red
6. Osmic Acid – black
7. Nile Blue Sulfate
PIGMENTS AND MINERALS 2. Giemsa Stain
1. Iron a. Bile pigments – Green
• Lillie’s Method b. Collagen, muscle, bone – Pale pink
o Ferric – Prussian Blue c. Microorganisms – Purplish-blue
o Ferrous – Turnbull’s Blue d. Starch granules, cellulose – Sky blue
• Perl’s Prussian Blue e. RBCs – Salmon pink
o Hemosiderin – Deep Blue f. Cytoplasm – light blue shades
• Gomori’s Prusian Blue – Bright Blue
• Leuco Patent Blue V 3. Wright-Giemsa or Jenner-Giemsa
o Hemoglobin Peroxidase – Dark a. Nucleus – Purple/blue
Blue b. Cytoplasm – Pink/blue
c. Eosinophils – Pink/red
2. Bile
• Modified Fouchet’s – Emerald to blue 4. May-Grunwald-Giemsa Stain
• Gmelin – Yellow to Green to Blue to 5. Perl’s Prussian blue stain – Iron stores
Purple to Red
• Schmorl’s – Dark Blue 6. Myeloperoxidase stain
a. Myeloid cells – Green to dark blue
3. Lipofuscin and Hemofuscin b. Eosinophils – Brown-black
• Gomori’s Aldehyde Fuchsin – Purple
7. Masson’s Trichrome Stain for GMA Plastic Bone
• Mallory’s Fuchsin – Red
Marrow Sections
• Schmorl’s – Dark Blue a. Osteoids seams – Red
b. Mineralized bone – Blue
4. Calcium/Bones
• Modified Von-Kossa – Black 8. Jones’ Impregnation – Black (Bone Marrow)
• Alizarin Red S – Reddish-orange
STAINING OF CONNECTIVE TISSUES
5. Copper 1. Reticulin Fibers
• Lindquist’s Modified Rhodanine a. Gomori’s Silver – Black
Technique – Red to Orange-red b. Gordon and Sweets’ – Black

6. Urate Crystals 2. Collagen

• Gomori’s Methenamine Silver – Black a. Van Gieson’s – Pink or Deep Red
b. Masson’s Trichrome - Blue
c. Gomori’s One-step – Green
STAINING OF BONE MARROW AND BLOOD d. Russell’s Modification - Yellow
ELEMENTS e. Mallory’s Aniline Blue - Blue
1. Wright’s Stain
a. RBCs – Yellowish-red 3. Amyloid
b. Granulocytes a. Azocarmine – Deep blue
i. Nucleus – Dark purple b. Congo Red – Red
ii. Granules – Reddish-lilac c. Crystal Violet – Purplish-Red
iii. Cytoplasm – Pale-pink d. Thioflavine
c. Eosinophils o Silver-blue Fluorescence at UV
i. Nucleus – Blue o Yellow Fluorescence at blue light
ii. Granules – Red to red-orange e. Lieb’s Crystal Violet – Purplish-Violet
iii. Cytoplasm – Blue
d. Basophils 4. Elastic Fibers
i. Nucleus – Purple to dark blue a. Weigert’s Resorcin-Fuchsin – Brown
ii. Granules – Very dark purple b. Vernhoeff’s – Black
e. Lymphocytes c. Vernhoeff-Van Gieson – Black
i. Nucleus – Dark purple d. Aldehyde Fuchsin – Deep Blue to Purple
ii. Cytoplasm – Sky blue e. Luna Staining – Purple
f. Platelets – Violet to purple o Mast Cells – Purple
f. Orcein – Dark Brown
g. Krajian’s – Bright Red

5. Fibrin
a. MSN – Yellow to Red to Blue
b. PTAH – Dark Blue
STAINING OF MUSCLE AND BONE
1. Muscle
a. Gomori’s Trichrome – Red
b. Frozen Section of Gomori’s Trichrome
i. Myofibrils – Green
ii. Intermyofibrillar –Bright Red
iii. Nemaline rods – Ragged Red
c. PTAH – Blue
2. Bone
a. Schmorl’s Picro-Thonin
i. Lacuna – Dark Brown-Black
ii. Canaliculi – Dark Brown-Black
iii. Bone Matrix – Yellow
b. Alizarin Red S – Orange-Red
c. Von Kossa– Black or Brown-Black
STAINING OF NERVOUS TISSUE
1. Cresyl Fast Violet
a. Nissl Bodies – purple-dark blue
b. Neurons – pale purple ble
2. Cajal’s Gold Sublimate
a. Astrocytes – bludh black
b. Nerve cells – red
3. Modified Holzer’s Method
a. Glial fibrils – blue
4. Myelin Sheath
a. Weigert-Pal – Blue Black
b. Luxol Fast Blue – Blue Black
c. Luxol Fast Blue-H&E – Blue/Green
d. Luxol Fast Blue-PAS-Hematoxylin –
Blue/Green
e. Weil’s Black
f. Baker’s Chromate-Acid Hematin – Dark
Blue
g. Swank & Davenport’s Marchi – Black
(Degenerating Myelin)
h. Methylene Blue-Azure II-Basic Fuchsin -
Blue
5. Microwave Modification of Bielschowsky’s
Technique
a. Axons – Brown to black
b. Cytoplasmic neutrofibrils – brown to
black
c. Neurofibrillary tangles – brown or black
d. Neuromelanin – black
6. Sevier-Munger
a. Neurites – black
b. Axons – black
c. Myelin sheath – light brown
d. Neuritic plaques – black
7. Modified PTAH
8. Bodian’s Stain
9. Golgi’s Silver staining
10. Modified Golgi method
11. Osmium Tetroxide
STAINING OF MICROORGANISMS
1. Gram stain
a. Counterstain – carbolfuchsin or neutral
red
b. GP – blue black; GN – red
2. Modified Brown-Brenn
a. Counterstain – basic fuchsin
b. GP – blue; GN – red
3. Gram-Twort Stain
a. Counterstain – neutral red and fast green
b. GP – blue-black; GN – pink-red
4. Ziehl-Neelsen Stain
a. Mycobacteria – red
5. Fite Stain
a. M. leprae – Bright red
b. Nocardia – Bright red
6. Microwave Auramine-Rhodamine
a. Acid-fast organisms – reddish-yellow
fluorescence
7. Toluidine Blue
a. Helicobacter – dark-blue
8. Cresyl violet
a. Helicobacter – blue-violet
9. Dierterle
a. L. pneumophila – brown to black
b. Spirochetes – brown to black
10. Warthin-Starry
a. Spirochetes – black
11. Steiner and Steiner Microwave
a. Spirochetes – dark brown to black
b. H. pylori – dark brown to black
c. L. pneumophila – dark brown to black
d. Other non-filamentous bacteria – dark
brown to black
12. Grocott Methenamine Silver Nitrate
a. Fungi cell wall – black
13. Lendrum’s Phloxine-Tatrazine
a. Viral inclusions – bright red
14. Orcein Method
a. HBsAg – brown-black
15. Giemsa
a. Protozoans – dark blue
b. Bacteria – blue