Histopath Lec staining Flashcards
(119 cards)
1
Q
is the process whereby tissue components are made visible in
microscopic sections by direct interaction with a dye or staining solution.
A
staining
2
Q
Most cells are ______________________, and therefore histological sections have to be stained in some way to make the cells visible.
A
colorless and transparent
2
Q
cells and tissues that are basic in character have greater affinity for _____ dyes
A
acidic dyes
2
Q
the purified form of a coloring agent or crude dye that is generally applied in an aqueous solution
A
histologic stain
2
Q
cells and tissues that are acidic in character have greater affinity for _____ dyes
A
basic dyes
3
Q
a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible
A
Mordant
3
Q
The great majority of routine histology is done with ________________ staining
A
hematoxylin and eosin (H&E)
3
Q
characteristic of Hematoxylin why it is widely used in most routine histology (3)
A
Quick, Cheap, Informative
3
Q
______________ is poorly permeable to most staining solutions and should therefore
be removed from the section prior to staining
A
Paraffin wax
3
Q
H&E stain involves the use of two contrasting stains, e.g., hematoxylin which stains the ___________, and eosin which brings out the
_______________ of the cell and the tissue’s architecture
A
nuclear detail, cytoplasmic detail
3
Q
Mordant for Ehrlich’s Hematoxylin
A
Potassium Alum
3
Q
Method of Mordanting wherein mordant is added followed by the dye
A
Pre-mordant
4
Q
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
A
Histological staining
4
Q
is the process of giving color to the sections by using aqueous or alcoholic dye solutions
A
Direct Staining
4
Q
is the process whereby the action of the dye is intensified by adding another agent or a MORDANT which serves as a link or bridge
between the tissue and the dye, to make the staining reaction possible
A
Indirect Staining
4
Q
Method of Mordanting wherein dye is added, followed by the mordant
A
Post-mordanting
4
Q
Method of Mordanting wherein it is mordant + dye added to the tissue
A
Metamordanting
4
Q
Mordant for Weigert’s Hematoxylin
A
Iron
4
Q
Mordanting technique that is not used for Histopath
A
Post-mordanting
4
Q
serves as a link or bridge
between the tissue and the dye, to make the staining reaction possible
A
Mordant
4
Q
is not essential to the chemical union of the tissue and the dye and merely accelerates the reaction
A
Accentuator
4
Q
Accentuator used for Loeffler’s methylene blue
A
Potassium Hydroxide
4
Q
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.
A
Regressive Staining
5
Q
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
A
Progressive Staining
5
Accentuator used for carbol thionine and carbol fuchsin
Phenol
5
if the primary stain used is a basic dye, the differentiation is carried out by an _________________ while _____________________ is used for differentiation after applying an acidic dye
acid solution, alkaline medium
5
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)
5
Staining that uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism.
Differential Staining
5
A mordant can act as a _____________________
differentiating agent
5
Staining that is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.
Metachromatic Staining
5
technique that 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
5
is reduced by argentaffin
cells, forming black deposits seen under
the microscope.
Ammoniacal silver
5
____________________ shows metachromasia more than methyl violet
Toluidine Blue
5
The dye that changes color when bound to tissue
Metachromasia
5
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
5
A metallic impregnating agent is different from a stain in that it is not _____________________________ on the surface as a precipitate or as
a reduction product in certain tissue components
absorbed by the tissue, but is held physically on
5
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
5
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. Common dyes used are lithium, carmine and India ink
INTRAVITAL STAINING
5
is a method of staining used in microscopy to examine
living cells that have been removed from an organism
SUPRAVITAL STAINING
5
Dye especially recommended for mitochondria.
Janus Green
5
probably the best vital dye.
Neutral Red
5
-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
5
Chromophore + Benzene
Chromogens
6
Provides color by absorbing specific wavelengths
Chromophore
6
is the corner stone of tissue-based
diagnosis
Hematoxylin and Eosin (H&E)
6
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
6
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
6
How is the deparaffinization typically done?
typically done with xylene (2 rounds, 1-2 mins each)
6
FACTORS INFLUENCING STAINING (5)
Dye affinity to tissue
Specimen geometry (thickness, surface, topology)
Target concentration
Reaction rate
Rate of stain loss (over- differentiation)
6
is the final step in histological processing and Ensures permanent adhesion of stained sections to the slide
Mounting
6
Drying methods after floatation and flattening
o Incubate at 37 deg C overnight
o Oven dry at 56-60 deg C for 2 hours, or Hot plate 45-55 deg C at 30-45 min
6
Drying method for CNS or delicate tissues
slower drying (e.g. 37 deg C for 24+ hours)
6
Properties of adhesive (3)
o Grease and dust free
o Non-reactive with stains
o Suitable for IHC (Immunohistochemistry), ISH (In-Situ Hybridization), and antigen retrieval
7
a substance which can be smeared on to the slides so that the sections stick well to the slides
ADHESIVES
8
most commonly use adhesive
Albumin
8
FORMULA
- Dried albumin 5 gm
- Sodium chloride 5 gm
- Dissolve in 100 cc. of Distilled Water and add crystals of thymol.
Dried Albumin
8
invaluable in cytology particularly for cytosine preparation
of proteinaceous or bloody material.
Aminopropyltriethoxysilane
8
is the most commonly used adhesive because it is very easy to make, is convenient, and is relatively inexpensive
Mayer’s egg albumin
9
used for mounting sections from distilled
water when the stains would be decolorized or removed by alcohol and xylene as would be the case with most of the fat stains (Sudan methods) or for metachromatic staining of amyloid
Aqueous mounting medium
9
has a low refractive index, is moderately transparent and evaporates easily, hence is good only for temporary mounting
Water
9
This is widely used as a section adhesive in immunohistochemistry.
PolyL-lysine
9
usually a syrupy fluid applied between the section and the coverslip after staining, setting the section firmly, preventing the movement of the coverslip.
mounting medium
10
the standard mounting medium used when dehydration
and clearing with xylene cannot be made (as in fat stains)
Glycerin jelly
10
Glycerin Jelly RI value
1.47
10
Farrant’s medium RI value
1.43
11
This is a very suitable semi-permanent mounting medium
with a refractive index of 1.46, sets quite hard, and will keep sections
mounted for years, especially if sealed on the edges with paraffin wax
Glycerin
12
recommended for mounting frozen sections from water
Brun’s Fluid
12
This medium is used for methylene blue-stained nerve preparations
and as a general purpose aqueous mountant. It is one of the most useful
aqueous mountants for fluorescent microscopy,
Apathy’s Medium
12
This gum arabic medium does not solidify upon storage and therefore
does not need to be heated before use. However, it takes a longer time to
harden and may therefore require ringing.
Farrant’s medium
12
Apathy’s Medium RI value
1.52
13
are used for
preparations that have been dehydrated and cleared in xylene or toluene, and
are recommended for majority of staining methods
RESINOUS MOUNTING MEDIA
13
It is a transparent, almost colorless oleoresin that adheres firmly to glass
and sets to a hard consistency without granulation
Canada Balsam
13
Canada Balsam RI value
1.524
13
is recommended for whole mounts and for thick sections
because it does not shrink much
Canada balsam
13
This is a resinous medium recommended for small tissue sections but not
for whole mounts because of shrinkage produced on drying; hence, it should be
used in excess amounts
DPX - (Dibutyl Phthalate and Xylene)
14
Canada Balsam is extracted from?
Abus Balsamea
14
is a synthetic resin mixture in xylene, available in a pale yellow or colorless solution.
XAM
14
DPX - (Dibutyl Phthalate and Xylene) RI value
1.532
14
is a synthetic resin which is soluble in xylene (it is
used as a 60% solution in xylene), and is generally preferred over D.P.X
Clarite
14
XAM RI value
1.52
14
Clarite RI value
1.544
14
is the process of sealing the margins of the cover-slip to prevent
the escape of fluid or semi-fluid mounts and evaporation of mountant, to fix the
coverslip in place, and to prevent sticking of the slides upon storage.
Ringing
15
dyes that are those obtained from plants and animals, previously utilized
for dyeing of wool and cotton
NATURAL DYES
15
It is by far the most valuable staining reagent used by the cytologist due to its powerful
nuclear and chromatin staining capacity, and its striking polychrome properties
which may be produced with proper differentiation
HEMATOXYLIN
15
4 Types of Natural Dyes
Hematoxylin
Cochineal dyes and its derivatives
Orcein
Saffron
15
HEMATOXYLIN is derived from
Hematoxylin Campechianum
15
The active coloring agent of Hematoxylin is ______________, which is formed by the oxidation of hematoxylin, a process known as “____________.”
hematin, ripening
15
accomplished by exposing the substance to air and
sunlight, thereby oxidizing hematoxylin
natural ripening
15
are substances that combine with the tissue and the staining
solution, forming a “bridge” that allows staining reaction to take place
Mordants
15
accelerated by adding strong oxidizing agents such as hydrogen peroxide, mercuric oxide, potassium
permanganate, sodium perborate or sodium iodate which converts hematoxylin
to hematin almost instantaneously by chemical oxidation
artificial ripening
16
are recommended for progressive staining of
tissues, and are usually counterstained with Eosin, Congo Red and Safranin
Alum hematoxylin stains
17
Alum hematoxylin stains example (2)
Ehrlich’s Solution
Harris’ Solution
17
Ehrlich’s Solution ripening agent
Sodium Iodate
17
are used only for differential or regressive staining, using Acid-Alcohol as a differentiating agent.
Iron hematoxylin compounds
18
is an old histologic dye extracted from the female cochineal
bug, which is treated with alum to produce the dye, carmine
COCHINEAL DYES
18
Harris’ Solution ripening agent
Mercuric Chloride
18
are utilized for the study of spermatogenesis
Copper hematoxylin solutions
18
Iron hematoxylin compounds example
Weigert’s Stain
19
COCHINEAL DYES are derived from
Coccus cacti
19
COCHINEAL DYES + picric acid
Picrocarmine
19
COCHINEAL DYES + Aluminum Chloride
Best’s carmine stain
20
Picrocarmine is used for ___________
Neuropathological studies
20
Best’s carmine stain is used for ____
the demonstration of
glycogen.
20
Synthetic dyes are also known as __________
coal tar dyes
20
Synthetic dyes are derived from
hydro-carbon benzene (C6H6)
20
is a vegetable dye extracted from certain lichens which are
normally colorless, but which, when treated with ammonia and exposed to air, produce blue or violet colors
ORCEIN
21
Synthetic dyes are collectively known as _____________
Aniline Dyes
21
are substances with definite atomic groupings and are
capable of producing visible colors
Chromophores
21
Dye that is recognized for its bright yellow- orange hue, distinct aroma, and unique taste
Saffron Dye
21
an auxiliary radical or substance which imparts to the compound the property of electrolytic dissociation, thereby altering the shade of the dye, enabling it to form salts with another compound, and ultimately retaining its color
Auxochromes
21
o Chemical ripening= Mercuric Chloride
o For routine nuclear staining, exfoliative cytology, and stain for sex chromosomes
o Oxidizing agent is very toxic and corrosive to automated staining machines
HARRIS’ Solution
21
process of converting the reddish-purple color of hematoxylin to a blue hue, which enhances contrast and improves visualization of tissue structures under the microscope
BLUEING
21
Saffron Dye is derived from
Crocus sativus
22
o Natural ripening (2 Months); Chemical ripening with Sodium Iodate
o Used for regressive staining, suitable for acidic tissues
Ehrlich’s Solution
22
o Natural ripening (4-6 months)
o Similar longevity with Ehrlich’s Hematoxylin
o Longer ripening
-DELAFIELD’S Hematoxylin
22
o Chemical ripening= Alcoholic Iodine
o Used after Celestine Blue
COLE’S Hematoxylin
22
o Chemical ripening= Sodium Iodate
o Short staining time
o Short storage
MAYER’s Hematoxylin
22
o Chemical ripening= Potassium Iodate
o Will stain mucins; prevents formation of surface precipitate with Ethylene Glycol (Ethanediol)
o Sensitive to acids; may stain adhesive and glass slide
GILL’S Hematoxylin
22
o Chemical ripening= Potassium Iodate
o Short Staining time (Progressive staining)
o Does not stain cytoplasmic organelles
CARAZZI’S Hematoxylin
