CHAPTER 16 PRINCIPLES OF STAINING Flashcards by Arriane Cyrel (MTI)

is the process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution.

Staining

How well did you know this?

Not at all

Perfectly

Certain parts of cells and tissues that are acidic in character (e.g.?) have greater affinity for basic dyes, while basic constituents (e.g.?) take more of the acid stains.

nucleus

cytoplasm

How well did you know this?

Not at all

Perfectly

Many dyes, however, require the use of a [?] - a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible.

mordant

How well did you know this?

Not at all

Perfectly

The great majority of routine histology is done with [?], because it is quick, cheap and informative.

hematoxylin and eosin (H&E) staining

How well did you know this?

Not at all

Perfectly

It involves the use of two contrasting stains, e.g., hematoxylin which stains the nuclear detail, and eosin which brings out the cytoplasmic detail of the cell and the tissue’s architecture.

hematoxylin and eosin (H&E) staining

How well did you know this?

Not at all

Perfectly

is poorly permeable to most staining solutions and should therefore be removed from the section prior to staining.

Paraffin wax

How well did you know this?

Not at all

Perfectly

This is usually done by immersing the paraffin section in a solvent (e.g. xylene) two times, at [?] duration each, for sections up to [?] thick.

1-2 minutes

10 micron

How well did you know this?

Not at all

Perfectly

is not miscible with aqueous solutions and low graded alcohol, and should therefore be subsequently removed with absolute alcohol, followed by descending grades of alcohol to prevent damage and detachment of sections.

Xylene

How well did you know this?

Not at all

Perfectly

The alcohol is then finally replaced with water before actual staining of section is performed. Such procedure is the exact reverse of impregnation and may be summed up by the phrase

“Sections to Water”.

How well did you know this?

Not at all

Perfectly

If an alcoholic stain is to be used, there is no more need to replace the alcohol with water. After deparaffinization with xylene, the section is transferred to decreasing grades of alcohol, and in such instances, the term “?” is used, and the staining procedure is subsequently done unless the tissue has been fixed in mercuric chloride solution, in which case, the section is taken “to water”.

Sections to Alcohol

How well did you know this?

Not at all

Perfectly

Sections must be left in the oven for a minimum of [?] before they are finally stained to avoid such problems.

30 minutes

How well did you know this?

Not at all

Perfectly

is the process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.

Histological staining

How well did you know this?

Not at all

Perfectly

Micro-anatomic stains, bacterial stains and specific tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this category.

Histological staining

How well did you know this?

Not at all

Perfectly

is the process of giving color to the sections by using aqueous or alcoholic dye solutions.

Direct staining

How well did you know this?

Not at all

Perfectly

Only one dye is used, which is washed away after 30–60 seconds, prior to drying and examination.

Direct staining

How well did you know this?

Not at all

Perfectly

[?] is a classic example of a simple stain. This stain will color all cells blue, making them stand out against the bright background of the light microscope.

Methylene blue

How well did you know this?

Not at all

Perfectly

Indirect staining is the process whereby the action of the dye is intensified by adding another agent or a

MORDANT

How well did you know this?

Not at all

Perfectly

serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.

MORDANT

How well did you know this?

Not at all

Perfectly

combines with a dye to form a colored “lake”, which in turn combines with the tissue to form a “tissue- mordant-dye-complex” that is rendered insoluble in ordinary aqueous and alcoholic solvents.

MORDANT

How well did you know this?

Not at all

Perfectly

This allows subsequent counterstaining and dehydration to be carried out easily.

MORDANT

How well did you know this?

Not at all

Perfectly

It is an integral part of the staining reaction itself, without which no staining could possibly occur

MORDANT

How well did you know this?

Not at all

Perfectly

may be applied to the tissue before the stain, or it may be included as part of the staining technique, or it may be added to the dye solution itself.

MORDANT

How well did you know this?

Not at all

Perfectly

Examples of mordants are

potassium alum with hematoxylin in Ehrlich’s hematoxylin, and iron in Weigert’s hematoxylin.

How well did you know this?

Not at all

Perfectly

is not essential to the chemical union of the tissue and the dye.

ACCENTUATOR

How well did you know this?

Not at all

Perfectly

It does not participate in the staining reaction, but merely accelerates the reaction.

ACCENTUATOR

Examples of ACCENTUATOR are

potassium hydroxide in Loeffler's methylene blue and phenol in carbol thionine and carbol fuchsin.

is the process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.

PROGRESSIVE STAINING

Once the dye is taken up by the tissue, it is not washed or decolorized. The differentiation or distinction of tissue detail relies solely on the selective affinity of the dye for different cellular elements.

PROGRESSIVE STAINING

With this technique, the tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained.

REGRESSIVE STAINING

is the most common method utilized for microanatomical studies of tissues, using the regressive staining which consists of overstaining the nuclei, followed by removal of superfluous and excessive color of the tissue constituent by acid differentiation.

Routine Hematoxylin and Eosin (H&E) staining

is the selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.

DIFFERENTIATION (DECOLORIZATION)

uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism.

Differential Staining

This is usually done by washing the section in simple solution (e.g. water or alcohol), or by the use of acids and oxidizing agents.

Differential Staining

One commonly recognizable use of differential staining is the

Gram stain.

Gram staining uses two dyes: [?] (the counterstain) to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria.

Crystal violet and Fuchsin or Safranin

entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia).

METACHROMATIC STAINING

Tissue components combine with these dyes to form a different color from the surrounding tissue. This is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.

METACHROMATIC STAINING

is a process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria.

METALLIC IMPREGNATION

is the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis), or by staining of pre-existing cellular components (true vital staining), as in the staining of mitochondria by Janus green.

VITAL STAINING

is done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system.

INTRAVITAL STAINING

Common dyes used are lithium, carmine and India ink.

INTRAVITAL STAINING

Although methyl violets, of which crystal violet is one, do give metachromatic staining, they are not considered to be the most effective for the purpose. The azures or toluidine blue are more effective usually.

METACHROMATIC STAINING

is a method of staining used in microscopy to examine living cells that have been removed from an organism.

SUPRAVITAL STAINING

It differs from intravital staining, which is done by injecting or otherwise introducing the stain into the body.

SUPRAVITAL STAINING

New Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining

SUPRAVITAL STAINING

To achieve desired effects, the stains are used in very dilute solutions ranging from

1:5,000 to 1:50,000.

Common dyes used are:

1 Neutral red 2. Janus green 3. Trypan blue 4. Nile blue 5. Thionine 6. Toluidine blue

-probably the best vital dye.

1 Neutral red

-especially recommended for mitochondria.

2. Janus green

-one gram of dye is dissolved in 100 ml. of sterile distilled water to be used immediately;

3. Trypan blue

it is dangerous to allow the suspension to stand for more than one hour, because it is likely to become toxic to the cell.

3. Trypan blue

is the corner stone of tissue-based diagnosis.

Hematoxylin and Eosin (H&E) staining

The process stains thin tissue sections so that pathologists can visualize tissue morphology.

Hematoxylin and Eosin (H&E) staining

plays a significant role in tissue-based diagnosis by coloring otherwise transparent tissue sections, and allowing cell structures including the cytoplasm, nucleus, and organelles and extra-cellular components to be clearly visible under the microscope.

Routine H&E staining

Fixation: Most fixatives can be used except osmic acid solutions which inhibit hematoxylin.

ROUTINE H&E STAINING in Paraffin Embedded Section (Regressive Staining)

For tissues fixed with mercuric chloride, the staining time in hematoxylin should be increased slightly while duration of eosin staining should be reduced. The mercury should be removed using a [?] in [?] and rinsed in water. The iodine is then removed by placing the slide in [?] solution for [?] and washing it well in running water for [?].

0.5% solution of iodine 80 to 95% alcohol 3% sodium thiosulfate 1 to 5 minutes 3 to 5 minutes

Alternatively, mercury deposits may be removed after sections are hydrated, by immersing the sections in [?] for 5 minutes, followed by [?] and subsequently washing the section in water prior to staining.

Gram's or Lugol's iodine sodium thiosulfate

Staining may be prolonged for [?]-fixed tissues (e.g. Flemming's fluid), for tissues subjected to long acid decalcification, and after prolonged storage in acid formalin or 70% alcohol.

chromium and osmium

The following staining methods are commonly employed for frozen sections, the choice depending upon the personal preference of the pathologist and the type of tissue section to be stained.

1. Hematoxylin-Eosin method 2. Thionine method 3. Polychrome Methylene Blue method 4. Alcoholic Pinacyanol method (used also for supravital staining of mitochondria and primarily for color sensitization in photography)

It is somewhat less favored than regressive staining due to the difficulty of producing sufficiently intense progressive staining of cell structures without staining other parts, thereby resulting in diffused color and obscured details.

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

For convenience, reagents for this rapid H&E stain are generally arranged in sequence using a series of Coplin jars.

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

This method takes only 5-10 minutes and produces well-differentiated sections that are semi-permanent and can be stored.

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

The remaining portion of tissue must be kept for routine processing and are made for comparison with frozen sections.

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

Stains may be effectively removed from the skin by prompt topical application of [?], followed by rinsing with tap water.

0.5% acid alcohol

Paraffin ribbons containing air bubbles, torn or inadequately infiltrated sections are likely to float from the slide when deparaffinized and stained.

COLLODIONIZATION

also recommended for sections that will be subjected to strong alkaline or acid solutions and for tissues that contain glycogen for demonstration.

COLLODIONIZATION

is soluble in absolute alcohol, and will be removed if absolute alcohol is used in the final dehydration prior to clearing of stained sections. Instead, sections treated with 95% alcohol may be transferred to a mixture of equal parts of chloroform, absolute alcohol and xylene (C.A.X,) then treated with xylene and mounted in Xam.

Cellulose nitrate (celloidin)

Old, bleached or faded sections may be re-stained: the slide is usually immersed in xylene for [?], or gently heated until the mounting medium begins to bubble.

24 hours

It is placed in a [?] solution for [?], rinsed in tap water and subsequently immersed in [?]for [?] or until the section is decolorized. After washing it again in running tap water for another 5 minutes, the section may then be re-stained with the appropriate staining technique.

0.5 potassium permanganate 5-10 minutes 5% oxalic acid 5 minutes

is the process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance.

Chemical ions such as calcium, molecules such as bile pigments, and biopolymers such as cellulose, DNA and specific enzymes are among the tissue components that can be identified using

HISTOCHEMICAL STAINING (HISTOCHEMISTRY)

The staining techniques employed for histochemistry are also usually applied for staining of histologic structures. Examples of such type of stains are

Perl's Prussian blue reaction for hemoglobin, and Periodic Acid Schiff staining for carbohydrates.

is a combination of immunologic and histochemical techniques using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigens (e.g., proteins) and phenotypic markers under the microscope.