Stains methods - Stains and Staining Solutions

1. Natural dyes

2. Synthetic dyes

These stains are derived from dyes, which can be classified into two main categories:

Natural dyes

• Obtained from natural sources such as plants and insects.

• are those obtained from plants and animals, previously utilized for the dyeing of wool and cotton.

1. Cochineal dyes

2. Logwood dyes

3. Vegetable extracts

Examples of natural dyes:

Cochineal dyes

• Extracted from insects, it is mainly used in carmine stains.

• Among the oldest histological dyes, derived from the female cochineal insect (Coccus cacti)

• Known for their versatility in staining and can be modified with different reagents to serve specific histological purposes.

Logwood dyes

Derived from the heartwood of the logwood tree, which is the source of hematoxylin.

Vegetable extracts

Various plant-derived substances are used for staining.

Synthetic/ Artificial dyes

• These are chemically synthesized and commonly derived from aniline or coal tar dyes.

• Widely used to their consistency, availability, and variety of staining properties.

• These dyes play a crucial role in histological staining by imparting color to biological tissues, allowing for better visualization for microscopic structures.

1. Basic fuchsin

2. Methylene blue

3. Eosin and crystal violet

Example of Synthetic dyes:

1. Hematoxylin

2. Cochineal dyes

3. Orcein

4. Saffron

Among the most common natural dyes available are:

Hematoxylin

• A widely used staining reagent in histopathology

• This makes it one of the oldest and most valuable stains used in tissue processing.

• It is compatible with almost all fixatives and is a permanent stain

• it is not a true basic dye

nuclei with great contrast

Hematoxylin is considered the most valuable staining reagent because of its ability to stain?

hematin

• Active coloring agent of hematoxylin

  Staining properties of hematoxylin comes from its oxidized form

Natural ripening

occurs when the solution is exposed to sunlight for three to four months, allowing oxidation to take place gradually.

Artificial ripening

is a faster method, where oxidizing agents such as hydrogen peroxide, mercuric oxide, potassium permanganate, sodium perborate, or sodium iodate are added to speed up the process.

TRUE

TRUE or FALSE: Ripened hematoxylin is seldom used alone due to its inherent low affinity for the tissue itself.

1. alum

2. iron

3. chromium

4. copper salts

• Hematoxylin is most frequently used in combination with these

• act as mordants, catalyzing or forming links between the stain and the tissue.

Mordants

• are substances that combine with the tissue and staining solution, forming a bridge allowing staining reaction to occur

• act as intermediaries, ensuring that the stain adheres to the tissue properly

TRUE

TRUE or FALSE: Without a mordant, hematoxylin would not effectively stain cellular structures, leading to weak or uneven coloration.

tissue-mordant-hematoxylin linkage

Mordant such as aluminum or iron salts binds to the tissue, and then hematoxylin binds to the mordant, forming what linkage?

blue or purple

Aluminum mordant gives what hue?

black

iron mordants result in a what color of stain?

carmine

• When Cochineal dyes are combined with alum, itf

• is a strong chromatin and nuclear stain, making it useful for highlighting nuclear structures in tissue sections.

picrocarmine

• When Cochineal dyes is mixed with picric acid, it produces this

• a stain particularly valuable in neuropathological studies, where it helps differentiate various nervous system structures.

Hematoxylin canpechianum

Hematoxylin is a natural dye extracted from the core or heartwood of the Mexican tree. What is the name of this tree?

Coccus cacti

Cochineal dyes is derived from the female cochineal insect. What is this insect?

best carmine stain

• When cochineal dyes is treated with aluminum chloride it becomes this stain

• is specifically used for the demonstration of glycogen, making it essential in glycogen storage diseases and liver pathology.

Orcein

• A vegetable dye extracted from lichens in its colorless form.

• primarily used for the staining of elastic fibers, making it valuable in identifying elastic tissue in histological samples.

pH indicator

litmus has poor staining properties. So, instead of being used as a histological stain, it is primarily used as a?

blue or violet

Orcein when combined with ammonia and exposed to air, it develops this color, making it useful for histological staining

litmus

• Orcein when mixed with lime, soda, ammonia, and air, it transforms into this

• is commonly used in litmus paper to test pH levels.

Chromophores

• Chemical groups that are responsible for producing visible colors in dyes.

• Essential for a dye's ability to absorb and reflect light.

Chromogens

• Compounds that contain chromophores, giving them the potential to be colored substances.

• May not yet be capable of binding effectively to tissues. These are different from the dyes in that any color that they impart to the tissue is not permanent, and can, therefore, be easily removed

Auxochromes

• Auxiliary chemical groups that enhance the dye's ability to bind to tissues.

• Do not produce color themselves, but help retain the color in the tissue by modifying the dye's ability, pore solubility, and reactivity.

• An auxiliary radical or substance which imparts to the compound the property of electrolytic dissociation, thereby altering the shade of the dye, enabling it to form salts with another compound, and ultimately retaining its color

TRUE

TRUE or FALSE: The coloring property is attributed to the chromophore

TRUE

TRUE or FALSE: The dyeing property to the salt-forming is the auxochrome

1. Acid dyes

2. Basic dyes

3. Neutral dyes

Dyes may be classified into three groups:

Acid dyes

• Active coloring substance is found in the acid component and the inactive based is usually the sodium salt of a sulfonate of r0saniline

• Picric acid

• Trichloroacetic acid

• Chromium-fixed tissues

Examples of acid dyes:

Picric acid

• Example of an acid dye that has the ability to form salt with an alkali

• it can fix, differentiate, and stain tissue all by itself

1. Used as a counterstain to basic cytoplasmic stains.

2. It is also a component of Van Gieson’s stain, which is used for connective tissue.

3. It is also combined with crystal violet for fungal microscopy and functions as a fixative, decalcifying agent, and tissue softener.

Uses of Acid dyes:

acidophilic

Basic cell structures have an affinity for the acid dye ions and are regarded as?

Basic dyes

• The active coloring substance is found in a basic component that combines with the acid radical

Methylene blue

• An example of a basic dye

• functions as both a pH indicator and a bacterial stain.

Mercuric chloride and Formaldehyde

Tissue fixed with these usually favor staining with basic dye

1. Primarily used for nuclear staining because chromatin is acidic.

2. It is also commonly applied in bacterial staining in microbiology.

Uses of Basic dye:

basophilic

Acidic cell structures have an affinity for basic dye ions and are therefore regarded as?

Neutral dyes

• Formed by combining aqueous solutions of acid and basic dyes, capable of staining cytoplasm and nucleus simultaneously and differentially

• Insoluble or barely soluble in water

Ethyl alcohol or acetic acid fixed tissues

These readily take in both basic and acidic dye

1. Romanowsky

2. Giemsa

3. Irishman’s stain

Examples of neutral dye:

Irishman’s stain

A neutral dye for leukocyte differentiation

stain both the cytoplasm and the nuclei simultaneously, making them valuable for differential staining in blood smears and other histological applications.

Tissue affinity of Neutral dyes

1. Mainly used in hematology for leukocyte differentiation.

Uses of neutral dyes:

Found in the acid component; the inactive base is usually a sodium salt of a sulfonate.

Chromophore location of acid dyes

Found in the basic component, combined with an acid radical (sulfuric, acetic, hydrochloric acid).

Chromophore location of basic dyes

Formed by combining an acid and basic dye in an aqueous solution.

Chromophore location of neutral dyes:

Acidophilic structures are basic components, such as collagen and eosinophilic granules of leukocytes.

Tissue affinity of acid dyes:

Basophilic structures are acidic components such as chormatin, mucus, cartilage matrix.

Tissue affinity of basic dyes:

• Hematoxylin

• Eosin

Common staining solutions:

Hematoxylin and Eosin staining

is a widely used technique for examining thin tissue sections in histopathology.

blue

Hematoxylin stains cell nuclei what color?

pink or red

Eosin an acidic dye stain the cytoplasm and connective tissue in varying shades of?

1. Alum hematoxylin

2. Iron hematoxylin

3. Tungsten hematoxylin

4. Copper hematoxylin

Types of hematoxylin

Alum hematoxylin ( Aluminum Hematoxylin Solutions)

• Recommended for progressive staining tissues

• It can also be used for regressive staining

• After staining, cell nuclei appear blue to purple.

• It is commonly used in routine H&E staining because of its effectiveness as a nuclear stain, making cell nuclei more prominent and easier to analyze under the microscope.

blueing

• involves treating the tissues with a mildly alkaline solution to develop the desired hue.

• is essential to convert the initial soluble red color of hematoxylin in the nucleus into a stable, insoluble blue color.

• This ensures proper contrast and enhances nuclear detail in histological staining.

• The process relies on an alkaline pH, which causes the mordant dilate to reform within the tissue, making the stain more permanent and resistant to fading.

The blueing step

is a critical part of H&E staining, ensuring that nuclear structures appear distinct and properly stained for microscopic examination.

Scott's tap water substitute

A mixture of potassium carbonate, magnesium sulfate, and water, which helps maintain tissue integrity while achieving the blueing effect

Ammonia water

It is a simple and effective alkaline solution.

1. Ammonia water

2. Dilute lithium carbonate

3. Scott's tap water substitute

Common blueing solutions used include:

Iron Hematoxylin (Iron Hematoxylin Solutions)

• Used only for differential or regressive staining using acid-alcohol as a differentiating agent

• these stains are more resistant to acidic solutions, making them useful in special staining procedures.

• The stain gives a black or dark gray appearance to nuclei, providing strong contrast and fine nuclear detail.

• preferred for elastic fibers, enhancing their visibility in histological sections.

• It also stains mitochondria, allowing for better identification in cytoplasmic structures.

• It also highlights muscle striations, making it valuable in muscle tissue studies.

• Lastly, it is commonly used in cytology for detailed nuclear staining, particularly in delicate cellular preparations.

• ferric chloride ( ferric ammonium chloride)

• ferric ammonium sulfate

What does Iron hematoxylin used as mordant?

1. Weigert's hematoxylin

2. Heidenhain’s hematoxylin

Examples of iron hematoxylin stains include:

Tungsten Hematoxylin ( Phosphotungstic Acid Hematoxylin)

• Natural ripening of this solution is achieved with light and air, but will take some months to ripen

• Usually demonstrates structures in paraffin as well as celloidin and from sections

• Staining time: 12-24 hours

Mallory’s (PTAH) technique

• It uses phosphotungstic acid as a mordant and is ripened naturally or with potassium permanganate.

• It stains muscle fibers blue and collagen red-brown, making it useful for identifying muscle striations and fibrin.

• Staining is usually progressive

blue

In Mallory’s, Nuclei, fibrin, muscle striations, and myofibrils are colored?

orange red or brownish red to deep brick-red stain

In Mallory’s, Nuclei, collagen, bone and cartilage take in what color?

Copper hematoxylin stains

are used for spermatogenesis studies as it highlights the different stages of sperm cell development.

Eosin

• It is a red general cytoplasmic stain that combines with hemoglobin to give an orange color.

• It serves as a secondary stain, primarily used as a counterstain to hematoxylin in histological staining.

• It is an acidic dye, meaning it binds to basic or alkaline cell components, such as cytoplasm, muscle fibers, red blood cells, and extracellular matrix structures, staining them in various shades of pink to red

1. Eosin Y

2. Eosin B

3. Eosin ES (Ethyl eosin)

Eosin exists in three primary forms

Eosin Y

• “Yellowish”

• The most widely used type, giving a strong pink to red stain in routine H&E staining.

• It is readily soluble in water, less in alcohol

• Available in both aqueous and alcoholic solutions, showing a green yellow fluorescence especially alcohol form

Eosin B

• Has a slightly bluish tint, offering a different staining contrast.

Eosin ES (Ethyl Eosin)

A less common variant with unique staining properties

blue to blue black

H&E staining expected staining results:

• Nuclei

bright orange-red

H&E staining expected staining results:

• RBCs Eosinophilic granules Keratin

purplish pink

H&E staining expected staining results:

• Basophilic cytoplasm Plasma cells Osteoblast

Pink or light blue to dark blue

H&E staining expected staining results:

• Cartilage

purplish blue

H&E staining expected staining results:

• Calcium Calcified bone

pink

H&E staining expected staining results:

• Decalcified bone matrix Collagen Osteoid

deep pink

H&E staining expected staining results:

• Muscle fibers

dark blue

H&E staining expected staining results:

• Karyosome

pale pink

H&E staining expected staining results:

• Cytoplasm Proteins in edema fluid