STAINING (INCOMPLETE kay Chenney ang sumpay hahaha) Flashcards
Alkaline structures (cytoplasm) are stained by?
Acidic stains (Eosin)
Enables one to see and study the architectural pattern of the tissue and physical characteristics and structural relationships of tissues and their cells
Staining/Dyeing
Staining/Dyeing is made possible through?
- capillary osmosis
- solubility
- absorption and
- adsorption of stains or dyes by tissue
Acidic structures (nucleus) are stained by?
alkaline/basic stains (hematoxylin)
The process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution,
producing coloration of the active tissue component
Histological staining
Enumerate the stains used for Histological Staining
- Microanatomic stains
- Bacterial stains
- Specific tissue stains
Used to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm
Histological staining
The process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance
Histochemical staining or Histochemistry
A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under
the microscope, using a wide range of monoclonal, fluorescent-labeled or enzyme-labeled antibodies
Immunohistochemical staining or Immunohistochemistry
Enumerate the stains used for Histochemistry Staining
- Perl’s Prussian Blue (hemoglobin); demonstrates ferric iron (Fe3+) in
tissues - Periodic Acid Schiff (Carbohydrates)
Enumerate the Classifications of Staining
1) Histological staining
2) Histochemical staining
3) Immunohistochemical staining
It is the basis of histochemistry
Specific staining
It is accomplished by controlled, specific chemical reactions designed to give a final color (staining) at the site/location of the structure of the substances in the cells or tissues
Specific staining
Enumerate the stains used for Specific staining
- Perl’s Prussian blue reaction
- Periodic Acid Schiff technique
The action of the dye is intensified by some other agents
Indirect staining
The staining of tissue by means of simple alcoholic/aqueous
solution of the dye
Simple staining/ Direct staining
Enumerate the stains used for Simple staining/ Direct staining
Methylene Blue and Eosin
Substance which when taken up by the tissue, helps make the in return serving as a link or bridge to make the staining reaction possible.
Mordant
An integral part of the staining reaction itself, without which, no staining could possibly occur
Mordant
Combines with a dye forming a colored “lake” which combines with tissue to form an insoluble “tissue-mordant-dye complex
Mordant
Enumerate the stains used as a mordant
- Potassium Alum with Hematoxylin in Ehrlich’s hematoxylin
- Iron in Weigert’s hematoxylin
Chemical substances that does not participate but merely increases or heightens the color intensity, selectivity and crispness of the stain
Accentuator
Differ from mordants in that they do not bind or link the tissue to the dye
Accentuator
Enumerate the stains used as an Accentuator
- Potassium hydroxide (KOH) in Loeffler’s Methylene blue
- Phenol in carbol thionine and carbol fuchsin
Staining is continued in a definite sequence until the desired intensity of coloring of the different tissue elements is attained
Progressive staining
No washing/ differentiation/ decolorization in between is
required solely relies on the selective affinity of dyes for different cellular elements
Progressive staining
Enumerate the stains used in Progressive staining
Gill’s hematoxylin and Mayer’s hematoxylin
The tissues are overstained and the excess dye is then removed until the desired intensity is obtained
Regressive staining
What are the two principles of regressive staining
- Overstaining
- Washing/ Differentiation/ Decolorization
This refers to selective removal of excess stain from tissue
Washing/ Differentiation/ Decolorization
Enumerate the stains used for regressive staining
- Acid Fast Staining
- Gram Staining
- Harris hematoxylin
- Ehrlich’s hematoxylin
- Delafield’s hematoxylin
This is a slower staining process in which the tissue is left in the staining solution just long enough to reach the desired endpoint
Progressive staining
This is a more rapid staining process in which the tissue is deliberately over stained and then de-stained
Regressive staining
This method of staining rapidly diffuses over the entire cell
Regressive staining
This method of staining is slow and selective in staining of tissue components
Progressive staining
Entails the use of the specific dyes that stains tissues with a color that is different from that of the stain color itself
Metachromatic staining or Metachromasia
Metachromatic staining is particularly employed for staining of:
i. Cartilage
ii. Epithelial mucins
iii. Connective tissues
iv. Amyloid
v. Mast Cell granules
Basic dyes belonging to Thizine and Triphenylmethane groups
Metachromatic dye
Enumerate the stain used for Metachromatic staining
Cresyl Blue for reticulocytes
The application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated
Counterstaining
What stain is used for counterstaining?
Eosin — for the cytoplasm
The selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (Cytoplasmic Phagocytosis)
Vital staining
This is resistant to vital stains, and therefore is not demonstrated
Nucleus of the living cell
Demonstration of nuclear structures during vital staining suggests permeability of the membrane by the dye, signifying the?
Death of the cell
This vital stain is used to stain reticuloendothelial system
Trypan blue
True vital staining of mitochondria
Janus Green B
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 RES.
Intravital staining
What are the common dyes used for intravital staining?
- Lithium
- India ink
- Carmine
Used to stain living cells immediately after removal from the living body
Supravital staining
This is probably the best vital dye
Neutral red
Makes use of heavy metals which are precipitated with selectivity of certain cellular and tissue components
Metallic impregnation
What is the greatest application of metallic impregnation?
Greatest application in tissue from the CNS and for the demonstration of reticulin
Differs from staining because it consists of an opaque black particulate precipitate
Metallic impregnation
The most commonly used agent for impregnation
Silver nitrate
A stain for metallic impregnation that is used for demonstration of lipids
Osmium tetroxide
Are those obtained from plants and animals, previously utilized
for dyeing of wool and cotton
Natural dyes
The most important and the most commonly used for routine histologic studies
Hematoxylin
A vegetable dye extracted from lichens which are normally colorless, but when treated with ammonia and expose to air, produce blue or violet colors
Orcein
Mainly used for staining Elastic fibers
Orcein
Cochineal is an old histologic dye extracted from the?
Female cochineal bug (coccus cacti)
A plant with orange stigmas yielding a dye
Saffron
Enumerate the four natural dyes
- Hematoxylin
- Orcein
- Cochineal
- Saffron
Sometimes known as “coal tar dyes” since they were originally manufactured from substances that have been taken from coal tar
Synthetic dyes
Derived from the hydrocarbon benzene and are collectively known as aniline dyes
Synthetic dyes
A synthetic dye should consist two substances namely:
1) Chromophores
2) Auxochromes
A Greek word which means “color-bearers”
Chromophores
Substances capable of producing visible colors
Chromophores
Substances which impart to the compound property of electrolytic dissociation, thereby altering the shade of the dye, giving it the property of forming salts with another compound and ultimately retaining its color
Auxochromes
A Greek word which means “color increaser”
Auxochromes
For a chromogen to be a dye, it must be composed of an _________ and therefore have salt-forming properties, ultimately retaining its color
acid and a base
The coloring property is attributed to the __________ and the dyeing property to the salty forming __________,
chromophore and auxochrome, respectively
A process of selective removal of excess dye
Differentiation
If the dye is a basic one, differentiation is carried out by an?
Acid solution
An alkaline medium is used for differentiation after?
an acid dye
A specialized fixative used in frozen section which serves to
localize antigens and hydrolytic enzymes
Formol calcium
It is also used for preservation of lipids
Formol calcium
It is similar with H and E technique but the differentiation stage
is omitted
Modified H & E technique
H & E staining color reaction of: Cytoplasm & proteins in edema fluid
pale pink
H & E staining color reaction of: Cartilage
Light blue to Dark Blue
H & E staining color reaction of:
Decalcified bone
Osteoid
Collagen
Pink
H & E staining color reaction of: Muscle fiber
deep pink
H & E staining color reaction of:
RBC
Eosinophil granules
Keratin
Bright orange to Red
H & E staining color reaction of: Calcium & Calcified bone
Purplish blue
H & E staining color reaction of:
Plasma cells
Osteoblast
Basophilic cytoplasm
Purplish pink
H & E staining color reaction of: Karyosome
Dark blue
H & E staining color reaction of: Nucleus
Blue to blue-black
Hematoxylin is a natural dye that is extracted from the core of heartwood of the?
tree Haematoxylon campechianum
By far the most valuable staining reagent used by the cytologist due to its powerful nuclear and active coloring agent
Hematoxylin
Usually done by exposing the substance to air and sunlight, thereby oxidizing hematoxylin
Ripening
Ripening is a slow process which usually takes about ______? But it can be accelerated by adding ________
3-4 months; strong oxidizing agents
Without this the ripened hematoxylin is almost useless because of its inherent low affinity for the tissue itself
Mordant
The complex of stain and mordant is called a
“lake”
Enumerate the examples of Mordants
- Aluminum
- Chromium
- Iron
- Copper
- Tungsten/ Phospotungstic acid (PTAH)
- Ferric salt
This causes an intense blue-black appearance when used as a mordant
Ferric salt
This is the most commonly used mordant. Gives a blue lake appearance. And increases the selectivity for nuclei, especially if acid is added or is used as a differentiating agent
Aluminum
A red acid dye used for cytoplasm differentially
Eosin
Commonly used as a background or contrast stain because it gives a leasing and useful contrast to nuclear stains such as hematoxylin
Eosin
Enumerate the examples of Eosin
- Eosin Y (Most commonly used)
- Eosin B (Rarely used)
- Eosin S/ Ethyl Eosin (Rarely used)
Papanicolaou stain makes use of three stains. What are these stains
- Hematoxylin
- OG- 6 (orange-green)
- Eosin Azure
In papanicolaou stain, this is used for staining nuclear structures
Hematoxylin
In papanicolaou stain, this is used for staining cytoplasm of mature cells (ex Mature superficial cells)
OG- 6 (orange-green)
In papanicolaou stain, this is used for staining cytoplasm of immature cells (ex. Intermediate and parabasal cells)
Eosin Azure
Eosin azure is composed of?
Eosin, Bismarck brown, Lithium carbonate, PTA, Light green stain
(36,50,65)
What is omitted in the modified pap’s stain?
Bismarck brown dye
What are improved in the modified pap’s stain?
1) Sharpness of color
2) Brilliant staining reactions
In Modified H & E technique, what stage is omitted?
Differentiation stage
