STAINING Flashcards
a routine staining procedure used to stain tissue to reveal the cell types and structures (nucleus, cytoplasm and cell organelle) as well presence of microorganism specially bacteria.
H and E stain (Hematoxylin and Eosin)
stains used other H and E, this reveals particular tissue structure elements or microorganism.
Special stains-
The process of applying colored dyes on a section to study the architectural pattern of the tissue and physical characteristics of the cell as well its chemical components.
STAINING
stains used to identify proteins and DNA/RNA sequence
Advance stains-
demonstrate the specific constituents of tissue thru chemical reactions.
Histochemical staining
Classification of staining
Histological staining
Histochemical staining
Immunohistochemical staining
demonstrate the general appearance of cells and tissues by staining its nucleus and cytoplasm.
Histological staining
Example of Histological Staining
H and E stain
it permits the microscopic localization of a specific tissue substance.
Histochemical staining
Example of Histochemical staining
Periodic acid Schiff (PAS)
Combination of immunological and histochemical stain.
Immunohistochemical staining
Stain used to demonstrate carbohydrates
Periodic acid Schiff (PAS)
It uses monoclonal and polyclonal enzyme-labeled antibodies.
Immunohistochemical staining
Method of staining
Direct staining
Indirect staining
Progressive staining
Regressive staining
Metallic staining
Counter staining
Vital staining
Application of simple dye to stain the tissue in varying shades of colors.
DIRECT STAINING
Example of DIRECT STAINING
Methylene Blue
use of MORDANT to facilitate a particular staining method.
INDIRECT STAINING
Example of MORDANT INDIRECT STAINING
aluminum hematoxylin and iron hematoxylin
the use of ACCENTUATOR to improve either the selectivity or the intensity of stain
INDIRECT STAINING
serves as linked or bridge between dye and tissue
Mordant
accelerates the speed of staining reaction.
Accentuator
Example of ACCENTUATOR INDIRECT STAINING
E.g. Carbol fuchsin.
Stain applied to the tissue in strict sequence and for specific times. The stain is not washed out or decolorized because there is no overstaining of tissue constituents
PROGRESSIVE STAINING
Example of Progressive Stain
H and E stain for frozen section
Tissue is first overstained and then the excess stain is removed by decolorizer.
REGRESSIVE STAINING
stains that differentiate particular tissue components by staining them a different color to that of original dye.
METACHROMATIC STAINING
Example of METACHROMATIC STAINING
Bismarck brown
Example of REGRESSIVE STAINING
H and E stain
is the removal of washing out of the excess stain until the color is retained only in the tissue components to be
DIFFERENTIATION (DECOLORIZATION)
Example of COUNTER STAINING
Hematoxylin (nuclear stain) and Eosin Y (cytoplasmic stain).
Metachromatic staining phenomenon is known as
Metachromasia
The phenomenon is known as metachromasia.
METACHROMATIC STAINING
application of one or more dyes that by contrast will bring out difference between the various cells and tissues.
COUNTER STAINING
Structures demonstrated are opaque and black.
METALLIC IMPREGNATION/STAINING
deposition of heavy metals salts on around cells or tissue constituents.
METALLIC IMPREGNATION/STAINING
Staining of structures in living cells, either in the body (in vivo) or in a laboratory preparation (in vitro).
VITAL STAINING
injecting a dye into animal body producing colorization of certain cells.
INTRAVITAL STAINING( IN VIVO)
staining of living cells immediately after the removal from the body.
SUPRAVITAL STAINING ( IN VITRO)
removal of wax by immersing it to solvent. Because wax is poorly permeable to stains.
Deparaffinization
it is done graded alcohols or acetones from 70% to absolute alcohol (100%).
Dehydration
Process of staining of paraffin sections
Deparaffinization
Hydration
Staining
Dehydration
Clearing
Mounting
The most common stain applied for histological study is Hematoxylin and Eosin. Tissue is place in an acid alcohol (differentiation) before eosin.
Staining
is done with graded alcohols from higher concentration to lower concentration. Alcohol and acetone are miscible with xylene. First change is made to absolute alcohol or acetone followed by 90%, 70% alcohol and finally distilled water.
Hydration ( sections to alcohol)-
application of mounting medium (syrup fluid) between the stained section and cover slip.
Mounting
stained section is cleared with xylene. Since xylene is miscible to most mountants.
Clearing
Classification of Dye used in stains
Natural Dyes
Synthetic Dyes
Process of Staining of paraffin section (GIVE THE ORDER)
Deparaffinization
Xylene I
Xylene II
Absolute alcohol I
Absolute alcohol II
Hydration
90% alcohol
70% alcohol
Water
Hematoxylin
1% acid alcohol
Eosin
Dehydration
70% alcohol
90% alcohol
Absolute alcohol (100%)
Clearing
Xylene
Xylene
Mounting
Microscopic Examination
What are the natural Dyes
Hematoxylin
Cochineal dyes
Orcein
What are the synthetic Dyes
Acid Dyes
Basic Dyes
Neutral Dyes (amphoteric)
This is the most popular dye used as a nuclear stain.
HEMATOXYLIN
Hematoxylin from the greek word?
Blood Wood
Dye Obtained from plants and animals.
Natural dyes
It develops staining property after oxidation.
Must have a mordant to strengthen the staining capability.
HEMATOXYLIN
It is derived from the log tree (Hematoxylon Camphecianum) mainly found in Mexico.
HEMATOXYLIN
What is the mordant for HEMATOXYLIN
Aluminum or Iron
What are the Aluminum Hematoxylin Solution
Ehrlich’s hematoxylin
Harris Hematoxylin
Cole’s Hematoxylin
Mayer’s Hematoxylin
Hematoxylin dissolved in ethyl alcohol, with aluminum potassium, glycerin, d. h20 and glacial acetic acid.
For regressive staining.
Not suitable for frozen section.
Ehrlich’s hematoxylin
Hematoxylin dissolved in ethyl alcohol, with aluminum potassium,mercuric oxide, d. h20 and glacial acetic acid.
For regressive staining
Used for nuclear staining, exfoliative cytology and sex chromosome staining.
Harris hematoxylin
What are the Iron Hematoxylin
Weigert’s Hematoxylin
Heidenhain’s hematoxylin
Phosphotungstic acid Hematoxylin (PTAH)
Hematoxylin dissolved in alcohol with HCL and ferric chloride and d. h20.
Used for demonstrating muscle fibers and connective tissues.
Weigert’s hematoxylin
For demonstration of nuclear and cytoplasmic inclusions.
Heidenhain’s hematoxylin
3 forms of Eosin
Eosin Y (yellowish)- most common
Eosin B (red color)
Eosin S (ethyl eosin)
Mordant used is phosphotungstic acid
For paraffin, celloidin or frozen sections.
Phosphotungstic acid hematoxylin ( PTAH)
Examples of COCHINEAL DYES
Picrocarmine
Bert’s carmine
A red acid dye
Used as a counter stain after hematoxylin and before methylene blue.
For background stain
Eosin
It is a scarlet dye made from the ground bodies of cochineal beetles, treated with alum to produce a dye, CARMINE.
COCHINEAL DYES
Cochineal dye for neuropathological studies
Picrocarmine
Cochineal dye for glycogen demonstration
Bert’s carmine
Vegetable dye extract from lichens (orchella weeds)
Used for staining elastic fibers
ORCEIN
Most of these are in Aniline base and derived from coal tar.
Synthetic dyes
Example of Acid dyes that can be a counter stain for Van Gienson’s stain
Acid Fuchsin (magenta red)
Picric acid
The active coloring substance is in the acid component
Acid dyes
Cell structure that can take up acid dye
Collagen
Eosinophiliic granules of WBC
Mixture of picric acid and acid fucshin.
Introduce by Ira Van Gienson
Van Gienson’s stain
Van Gienson’s stain
Color of Collagen Fibers and muscle -
Other tissue components -
Red
Yellow
Active coloring component is in the basic components
Basic dyes
Example of Basic dyes
Methylene Blue
Cell structure with affinity to basic dye are
Chromatin
Mucus
Cartilage matrix
Combination of acid and basic dyes than can stain nucleus and cytoplasm simultaneously and differentially.
Neutral dye
Example of Neutral Dyes
Romanowsky Stain (methylene blue + eosin Y)
Giemsa Stain
used for parasite identification in peripheral blood smear.
Giemsa Stain
