PRINCIPLES OF STAINING Flashcards
• The process of applying dyes on the sections
STAINING
is the process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution
Staining
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
Staining
Made possible because of:______ of tissue and cells for dyes
Varying affinity
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
A_______ is the purified form of a coloring agent or crude dye that is generally applied in an aqueous solution
histologic stain
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
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
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
_______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
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
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
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
If an_____ is to be used, there is no more need to replace the alcohol with water
alcoholic stain
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
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
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
Sections must be left in the oven for a minimum of_____ minutes before they are finally stained to avoid such problems.
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CLASSIFICATION OF STAINING
According to purpose
Histological
Histochemical
Immunohistochemical
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
Micro-anatomic stains, bacterial stains and specific tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this category.
Histological staining
METHODS OF STAINING (10)
- Direct Staining
- Indirect Staining
- Progressive STaining
- Regressive Staining
- Microanatomical Staining
- Metachromatic Staining
- Counterstaining
- Metallic Impregnation
- Vital Staining
- Intravital Staining
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
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
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
