Staining Solutions (lab)

HEMATOXYLIN

From the core or the heartwood of Hematoxylin campechianum (Mexican Tree)

Hematin

Active coloring agent is _______ (from the oxidation of the Hematoxylin or Ripening)

Natural Ripening

expose the extract to air or sunlight, takes around 3-4 months

Artificial Ripening

Adding oxidizing agents (Hydrogen Peroxide, Mercuric Oxide, Potassium Permanganate, Sodium Perborate or Sodium Iodate)

Ripened Hematoxylin

needs mordants (Alum, Iron, Chromium and Copper Salts)

COCHINEAL DYES

An old histologic dye from the Female Cochineal bug (Coccus Cacti)

COCHINEAL DYES

• Treated with Alum to produce the dye Carmine

• Powerful Chromatin and Nuclear stain for Fresh Material and Smear Preparation

Add Picric Acid (Picrocarmine)

used in Neuropathological studies

Add Aluminum Chloride (Best Carmine Stain)

for Glycogen

ORCEIN

Vegetable dye from certain lichens which are normally colorless.

blue to violet

ORCEIN- Treatment with Ammonia and exposure to air produces

ORCEIN

Soluble with Alkali, for Elastic Fibers

Litmus

came from lichens, treated with Lime and Soda,

Exposed to ammonia and air. Used as an indicator

ARTIFICIAL/ SYNTHETIC DYES

those came from substances taken from Coal Tar. Derived from Hydrocarbon Benzene; known as Aniline Dye.

CHROMOPHORES

substances with definite atomic groupings, capable for visible colors.

Chromogens

Simple Benzene compounds which contain these substances are called

CHROMOPHORES

The colors are not permanent, it requires the addition of an Auxochrome.

AUXOCHROME

auxiliary radical that imparts electrolytic dissociation, alters the shade of the dye, forms salts with another compound and ultimately retains the color.

ACID DYE

• Coloring agent is on the acidic component

• Basic Cell structures have affinity to Acidic Stains

ACID DYE

Examples: Acid Fuchsin, Picric Acid

Picric Acid

can be used as a fixative, can differentiate or stain tissue by itself. Decalcifying agent and a tissue softener

BASIC DYE

• Coloring agent is on the basic component

• Acidic Structures have affinity to Basic Stains

BASIC DYE

Examples: Methylene Blue (can be used as an indicator and a dye) (For Bacterial Staining)

NEUTRAL DYE

• Formed by combining aqueous solutions of acidic and basic dyes

• Can stain Cytoplasm and Nucleus simultaneously and differentially.

NEUTRAL DYE

• Insoluble to barely soluble to water

• Soluble in Alcohol

NEUTRAL DYE

Examples: Romanowsky Dyes in Hematology, Giemsa’s Stain. Irishman’s Stain for Leukocytes differentiation

HEMATOXYLIN

for routine histologic studies, needs the mordant Iron and Alum.

ALUMINUM HEMATOXYLIN

• For Progressive staining and Regressive Staining

• Aluminum salts give a blue lake

EHRLICH’S HEMATOXYLIN

• Regressive Staining

• Cartilage, Cement lines of bones – intensely blue.

EHRLICH’S HEMATOXYLIN

• Not ideal for Frozen Sections

• Suitable for tissues undergone acid decalcification

EHRLICH’S HEMATOXYLIN

Ripened by Sodium Iodate

HARRIS HEMATOXYLIN

Good Regressive stain; Routine Nuclear Staining

HARRIS HEMATOXYLIN

• Precipitation in staining solution – deterioration in the nuclear staining property

• Filtration is needed and contact time is increased.

HARRIS HEMATOXYLIN

Ripened by Mercuric Chloride

COLE’S HEMATOXYLIN

Used in sequence with Celestine Blue

COLE’S HEMATOXYLIN

Artificially ripened with Alcoholic Iodine solution

CARAZZI’S HEMATOXYLIN

ripened by Potassium Iodide, Frozen Sections

IRON HEMATOXYLIN

For differential and regressive staining; Uses Acid Alcohol as differentiating agent

IRON HEMATOXYLIN

Stain becomes more selective for nuclei if acid or an excess of Ferric salts are added.

WEIGERT’S HEMATOXYLIN

Standard Iron Hematoxylin (Muscle fibers and Connective Tissues)

HEIDENHAIN’S HEMATOXYLIN

Cytological staining; Regressive Staining

LOYEZ HEMATOXYLIN

frozen sections

VERHOEFF HEMATOXYLIN

Elastic Fibers stained Black.

PHOSPHOTUNGSTIC ACID HEMATOXYLIN

• Mordant used is 1% Aqueous Phosphotungstic Acid

• Progressive Stain

PHOSPHOTUNGSTIC ACID HEMATOXYLIN

Oxidizer used is Potassium Permanganate

LEAD HEMATOXYLIN

for granules of endocrine cells of the alimentary tract

Eosin

differentiates Connective tissues and Cytoplasm

Eosin

Used as a background stain because it gives a pleasing and colorful contrast to nuclear stains. (in Chromate and Picric Acid fixed tissues)

Eosin

Used as a counterstain after hematoxylin and before methylene blue

EOSIN Y

• Most commonly used. Soluble in Water, less soluble in alcohol.

• Available in Aqueous and Alcoholic solutions.

• Green Yellow Fluorescence especially in alcoholic medium.

EOSIN B

Or Erythrosin B; showing deeper red color

EOSIN S

Or Ethyl Eosin, Alcohol soluble

5% AQUEOUS EOSIN Y

Eosin Y dissolved in water. With Thymol crystal to avoid fungal growth

ALCOHOLIC EOSIN

• Eosin Y dissolved in water. Add Alcohol. Add Glacial Acetic Acid

• Combining Eosin Y and Phloxine B – produces a cytoplasmic stain.

OIL SOLUBLE DYES (LYSOCHROMES)

• not a real dye because they do not have the auxochrome groups.

• Give colors to lipids because they are more soluble to lipid medium of the tissue

SUDAN BLACK

• Most sensitive of all oil soluble dyes. Has more greater affinity for phospholipids than other lysochromes

• Colors Neutral lipids like Triglycerides

SUDAN BLACK

Staining ability relies on the dye’s concentration, temperature and physical state of the fats

SUDAN B

Stains phospholipids and neutral fats. Doesn’t stain crystalline cholesterol and free fatty acids tend to be soluble in the ethanolic dye bath

SUDAN IV

Or Scharlach R. Has no secondary amino group. Doesn’t color phospholipids or the fine lipid droplets.

Add Benzoic Acid

• intensifies Fat and prevents rapid deterioration of the solution. Recommended for staining triglycerides

• Giving them a deep and intense red color

SUDAN III

First Sudan Dye introduced into histochemistry.

SUDAN III

Fat Soluble, Good as a fat stain for Central Nervous System tissue.