PRINCIPLES OF STAINING Flashcards by Kyra Catedral

• The process of applying dyes on the sections

STAINING

How well did you know this?

Not at all

Perfectly

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

Staining

To see and study the_____ of the tissue, the _______of the cells, and the ______of the tissue and their cells

architectural pattern

physical characteristics

structural relationship

How well did you know this?

Not at all

Perfectly

Staining

Made possible because of:______ of tissue and cells for dyes

Varying affinity

How well did you know this?

Not at all

Perfectly

Most cells are______ and______, and therefore histological sections have to be stained in some way to make the cells visible. The same is true of components of the extracellular matrix.

colorless and transparent

How well did you know this?

Not at all

Perfectly

A_______ is the purified form of a coloring agent or crude dye that is generally applied in an aqueous solution

histologic stain

How well did you know this?

Not at all

Perfectly

Certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater affinity for______ dyes, while basic constituents (e.g. cytoplasm) take more of the____ stains.

Individual variation of the tissue constituents regarding these properties will consequently produce variation in colors under the microscope.

basic

acid

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.

When excess dye solution is washed away, the_____ stain remains

mordant

mordanted

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.

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

hematoxylin and eosin (H&E) staining

hematoxylin

eosin

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.

This is usually done by immersing the section in a solvent (e.g.______) two times, at 1-2 minutes duration each, for sections up to____ micron thick.

Paraffin wax

xylene

10um

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______ 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”.

alcohol

How well did you know this?

Not at all

Perfectly

If drying is not complete, the section (or part of it), especially from_____ and_____ tissue, may become detached from the slide during the process of staining, usually after adding the acid differentiator.

bone and nervous

How well did you know this?

Not at all

Perfectly

If an_____ is to be used, there is no more need to replace the alcohol with water

alcoholic stain

How well did you know this?

Not at all

Perfectly

After staining, the section is again______ with increasing grades of alcohol and cleared with two changes of_____ to prepare the section for mounting, since most mountants are miscible in _____.

dehydrated

xylene

How well did you know this?

Not at all

Perfectly

True or False

The stained section may be left in xylene for an indefinite period of time until it is finally mounted on the slide.

True

How well did you know this?

Not at all

Perfectly

True or False

The section should not be allowed to stay in alcohol for a Jong time because many stains are usually removed by prolonged immersion in alcohol.

True

How well did you know this?

Not at all

Perfectly

Sections must be left in the oven for a minimum of_____ minutes before they are finally stained to avoid such problems.

How well did you know this?

Not at all

Perfectly

CLASSIFICATION OF STAINING
According to purpose

Histological
Histochemical
Immunohistochemical

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

METHODS OF STAINING (10)

Direct Staining
Indirect Staining
Progressive STaining
Regressive Staining
Microanatomical Staining
Metachromatic Staining
Counterstaining
Metallic Impregnation
Vital Staining
Intravital Staining

How well did you know this?

Not at all

Perfectly

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

Histochemical staining

How well did you know this?

Not at all

Perfectly

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 techniques

How well did you know this?

Not at all

Perfectly

The staining techniques employed for ______ are also usually applied for staining of histologic structures. Examples of such type of stains are_______ reaction for hemoglobin, and_______ staining for carbohydrates.

histochemistry Perl's Prussian blue Periodic Acid Schiff

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

IMMUNOHISTOCHEMICAL (IHC) STAINING

is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors, in the localization of biomarkers and differentially expressed proteins in different parts of a biological tissue, and in the detection of specific molecular markers that are characteristic of particular cellular events such as proliferation or cell death (apoptosis).

Immunohistochemical staining

Visualizing an antibody-antigen interaction can be accomplished in a number of ways.

Immunohistochemical staining

are used to label defined antigens with monoclonal and polyclonal antibodies.

Immunohistochemical staining techniques

Commercially produced antibodies most frequently originate from____, and less frequently from rabbits.

mice

The current recommendation for immunohistochemical techniques is a maximum of______ solution and, for some antibodies, fixation time can be up to a maximum of____ Microwave-based fixation of tissue in formaldehyde may have an adverse effect on immunohistochemical staining.

4% neutral buffered formaldehyde 48 h

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

Direct staining

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

simple (or direct) staining

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

Methylene blue

is the process whereby the action of the dye is intensified by adding another agent or a_____ which serves as a link or bridge between the tissue and the dye, to make the staining reaction possible

Indirect staining MORDANT

By itself, the dye may stain only weakly, if at all. The mordant combines with a dye to form a colored "_____", which in turn combines with the tissue to form a "______" that is rendered insoluble in ordinary aqueous and alcoholic solvents.

lake tissue-mordant-dye-complex

Lake

Mordant + Dye

Lake + tissue

Tissue-mordant-dye complex

A______ 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

Examples of mordants are: __________ in Ehrlich's hematoxylin, and______ in Weigert's hematoxylin.

potassium alum with hematoxylin iron

is not essential to the chemical union of the tissue and the dye. It does not participate in the staining reaction, but merely accelerates the reaction.

ACCENTUATOR

Accentuator Examples are: _________ in Loeffler's methylene blue and_________ in carbol thionine and carbol fuchsin.

potassium hydroxide phenol

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

Routine Hematoxylin and Eosin (H&E) staining is the most common method utilized for microanatomical studies of tissues, using the______ staining which consists of overstaining the nuclei, followed by removal of superfluous and excessive color of the tissue constituent by______

regressive acid differentiation

Regressive staining Example:

Gram's Stain Acid fast staining

3 CLASSES OF DIFFERENTIATORS

Acid differentiator Oxidizing differentiator Mordant differentiator

Forms a soluble salt with the metal so that the latter is dissolved out

Acid Differentiator

Acid Differentiator examples

HCI HAc

Oxidizes the dye to a colorless substance

Oxidizing Differentiator

Oxidizing differentiator examples

K ferricyanide K permanganate

If the primary stain is basic, the decolorizer is acidic, and vice-versa Alcohol acts as a differentiator for both basic and acidic dye Differentiation is also done to retain faded slide

Mordant Differentiator

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)

A staining procedure that differentiates or distinguishes between types of bacteria is termed as a_______

differential staining technique

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

Differential Staining

In general, if the primary stain used is a basic dye, the differentiation is carried out by an acid solution, while alkaline medium is used for differentiation after applying an acidic dye.

Differential Staining

acts as a differentiator for both basic and acidic dyes, probably by simply dissolving out the excess dye.

Alcohol

Differential staining is also used to detect abnormalities in the proportion of different white blood cells in the blood. The process or results are called a________

WBC differential

T or F A mordant can act as a differentiating agent

True

Mordants such as_____ can also oxidize hematoxylin to a soluble, colorless compound, so that the tissue component becomes decolorized

iron alum

T or F if a section that has been stained by a mordant dye is allowed to remain in a differentiating agent such as 1 to 2% alcohol, all the dye will be removed

True

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 Fuchsin or Safranin

Most dyes stain tissues______, i.e., in color shades that are similar to the color of the dye itself.

orthochromatically

….technique 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 (______).

Metachromatic staining metachromasia

Tissue components combine with these dyes to form a different color from the surrounding tissue.

Metachromatic staining

This is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.

Metachromatic staining

Although______, of which crystal violet is one, do give ***metachromatic staining***, they are not considered to be the most effective for the purpose. The____ or _____ are more effective usually.

methyl violets azures or toluidine blue

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

Metallic Impregnation Specific tissue elements are demonstrated, not by stains, but by colorless solutions of ______which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria.

metallic salts

Metallic impregnation _______, for example, is reduced by argentaffin cells (e.g. in melanin and intestinal glands), forming black deposits seen under the microscope.

Ammoniacal silver

A_______ is different from a stain in that it is not absorbed by the tissue, but is held physically on the surface as a precipitate or as a reduction product in certain tissue components

metallic impregnating agent

metallic impregnating agent The most valuable metals for this purpose are

gold (gold chloride) silver (silver nitrate)

….are potentially explosive, care should be taken to prepare all solutions in clean containers just before use, and silvered glassware should be avoided. Flexible plastic containers may be used instead. Solutions should never be exposed to sunlight if explosion is to be avoided, and all unused reagents should be immediately inactivated by ____ or ____solution and discarded.

ammoniacal silver solutions sodium chloride or dilute hydrochloric acid

T or F The use of metallic instruments should be avoided when handling sections for metallic impregnation.

True

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

are excluded by the living cells but taken up by the already dead cells as in the _____ of reticulo-endothelial system with trypan blue, or propidium iodide for eukaryotic cells.

Vital stains

Vital stains are excluded by the living cells but taken up by the already dead cells as in the vital staining of reticulo-endothelial system with_____, or _______ for eukaryotic cells.

trypan blue propidium iodide

The nucleus of a living cell is resistant to_______, and therefore is not demonstrated. In fact, demonstration of nuclear structures during _____suggests permeability of the membrane of the dye, signifying the death of the cell.

vital stains

….of living cells 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

Intravital staining Common dyes used are

lithium India ink carmine

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

Supravital staining

Those that enter and stain living cells are called ________ However, these stains are eventually toxic to the organism, some more so than others.

Supravital staining

Examples of supravital stains

New Methylene Blue Brilliant Cresyl Blue for reticulocyte staining Neutral Red Janus Green Trypan Blue

• Stains living cells immediately after removing from the living body

SUPRAVITAL STAINING

-probably the best vital dye.

Neutral red

-especially recommended for mitochondria

Janus green

-one gram of dye is dissolved in 100 ml. of sterile distilled water to be used immediately; it is dangerous to allow the suspension to stand for more than one hour, because it is likely to become toxic to the cell.

Trypan blue

• Application of a different color or stain to provide contrast and background staining

COUNTERSTAINING

Cytoplasmic Counterstaining

Eosin Y Picric Acid Lissamine Green

• Nuclear Counterstaining

Neutral Red Hematoxylin Safranin O

• General relationship of tissues and cells

MICROANATOMICAL STAINING

- structures found in the cytoplasm and nucleus - bacterial morphology

• Cytoplasmic Staining • Negative Staining