Staining Flashcards
The process of applying dyes on the sections to see and study the __________ of the tissue and _______ _______ of the cells
Staining
architectural pattern
physical characteristics
FACTORS INFLUENCING DYE BINDING
pH of the sol’n
INCREASE temp
INCREASE conc. of dye molecules
[+] other salts
Fixative type
Staining of Tissues can be Classified into 3 Major Groups
Histological staining
Histochemical staining (Histochemistry)
Immunohistochemical staining
The process whereby tissue constituents are demonstrated in sections by direct interaction with a dye
Histological staining/Microanatomical staining
used to demonstrate the general relationship of tissues and cells with differentiation of _________
Histological staining/Microanatomical staining
nucleus cytoplasm
The process whereby various constituents if tissues are studied through chemical reactions that will permit _______ localization of a specific tissue substance
Histochemical staining/Histochemistry
microscopic
Example of dyes whereby various constituents if tissues are studied through CHEMICAL RXN that will permit MICROSCOPIC LOCALIZATION of a SPECIFIC tissue substance
Per’s prussian blue rxn for hemoglobin
Periodic Acid Shiff staining for carbohydrates
__________
allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of ff Antibodies: [4]
Immunohistochemistry
Polyclonal
Monoclonal
Fluorescent labeled
Enzyme-labeled
The process by W/O sections are stained with simple Aqueous or Alcoholic solutions of the dye
Direct Staining
The process by which sections are stained with an alcoholic solutions of dye with the use of a mordant/accentuator
Indirect Staining
A substance that serves as a link or bridge to make the staining reaction possible.
What do we call the complex
Mordant
tissue-mordant-dye complex
Stains that serves as as a link or bridge to make the staining reaction possible.
Potassium Alum w/ Hematoxylin in Erlich’s hematoxylin
Iron in Weidgert’s hematoxylin
___________
A substance that does not participate in staining reaction, but merely accelerate or hasten the speed of staining reaction by increasing the [2]
Accentuator
Staining power
Dye Selectivity
Accentuators that does not participate in staining reaction, but merely accelerate or hasten the speed of staining reaction by increasing the staining power and selectivity of the dye
Potassium Hydroxide in Loeffler’s Methylene Blue
Phenol in Cabon Thionin
Carbol Fuschin
➔ Stained in definitive sequence
➔ Not washed / decolorized
Progressive staining
➔ Differentiation of tissue details relies solely on the
SELECTIVE AFFINITY of dyes for different cellular elements
Progressive staining
A progressive stain that relies solely on the
SELECTIVE AFFINITY of dyes for different cellular elements
Papanicolau smear
The tissue is first over stained to obliterate the cellular details, and then removed or decolorized from unwanted parts of the tissue
Regressive staining
Selective removal of excess stain from the tissue during regressive
Differentiation/Decolorization
➔ Entails the use of specific dyes which differentiate particular substance by staining them with a color that is different from that of the stain itself.
Metachromatic staining
Metachromatic dyes are basic dyes belonging to [2]
Thizine group
Triphenylmethane group
Metachromatic staining are designated for these locations [5]
Cartilage
CT
Epithelial mucins
Mast cell granules
Amyloid
Basic dyes belonging to the thizine and triphenylmethane groups [9]
Azure A,B,C
Basic fuschin
Bismarck brown
Cresyl Blue
Methyl Violet
Methylene Blue
Safranin
Thionine
Toluidine blue
Used to demonstrate the general relationship of tissues and cells with general differentiation of nucleus and cytoplasm without necessarily emphasizing the inclusion bodies
Microanatomical staining
Specific tissue elements are demonstrated by colorless solutions of metallic salts.
Metallic Impregnation
A stain example where specific tissue elements are demonstrated by colorless solutions of metallic salts.
Ammoniacal Silver – produces black
_________
➔ Different from a stain. It is absorbed by the tissue, but held [chemically/physically] on the surface as a ________ or as a _______ product in certain tissue components.
➔ [COLORS]
Metallic impregnation
physically
Precipitate
Reduction product
Gold/Silver
_____________
A selective staining of living cell constituents, demonstrating cytoplasmic structures by _______ of the dye particle
Vital staining
phagocytosis
Examples of vital stains [+designation]
Typan Blue – RE system
True Vitals Staining- mitochondria
Staining of living cells is done by injecting the dye into any parts of the animal body
Intravital staining
Stains/Dyes that stains living cells by injecting the dye into any parts of the animal body
Lithium
India ink
Carmine
Used to stain living cells immediately after the removal from the living body
Supravital staining
Common Vital Stain/Dyes
Neutral Red
Janus Green
Trypan Blue
Nile Blue
Thionine
Toluidine Blue
Best vital dye
Neutral red
Recommended for mitochondria
Janus Green
Toxin when stand for more than 1 hour
Trypan Blue
Enumerate all methods of staining [11]
Direct
Indirect
Progressive
Regressive
Differentiation/Decolorization
Metachromatic staining
Microanatomical staining
Metallic Impregnation
Vital
Intravital
Supravital
Categories of Staining [2]
Natural
Synthetic
Obtained from plants and animals previously dyeing of wool and cotton
Natural dyes
Examples of natural dyes that were obtained from plants and animals previously dyeing of wool and cotton
Cochineal dyes [+derivatices]
Orcein
Saffron
Hematoxylin
dye recommended for mitochondria
Janus green
becomes toxin when stand for more than 1 hr
Trypan blue
Natural dye derived by either extraction from the heartwood of a _______ tree
➔ Hematoxylin ________ (B.E.Q.)
Hematoxylin
mexican
campechianum
Considered as the valuable staining reagent
by cytologist
Hematoxylin
➔ Has powerful nuclear and chromatin staining
capacity
Hematoxylin
➔ Has striking polychrome properties
Hematoxylin
_________
Old histologic dye derived from extract female _____ [other term] -
(B.E.Q)
Cochineal dyes
cochineal bug
coccus cacti
Cochineal dyes are treated with _______ or produce a dye known as _______.
alum
carmine
extensively used as powerful chromatin and nuclear stain for fresh material and smear preparation
Cochineal dyes
_________
Vegetable dye extracted from certain ______ (B.E.Q.)
Orcein
lichens
Normally colorless but when treated with ______ and expose to _____ produce [2 colors]
ammonia
air
blue/violet
a dye that is weak acid & soluble in alkali
Orcein
Mainly used for elastic fibers
Orcein
_________
Derived from the fried stigmata of ________ (B.E.Q.)
Saffron
croccus sativus
Known as “coal tar dyes” as they are manufactured from substance that have been taken from coal tar
Synthetic dyes
Synthetic dyes are derived from
hydrocarbon benzene
Synthetic dyes are collectively known as
Aniline dyes
Synthetic dyes are classified into 3 groups
Acid dyes
Basic dyes
Natural dyes
Coloring substance are found in the acid component
an example of acid dyes that is outstanding in the sense that it is the only substance so far that can fix the stain tissues all by itself
Picric acid
Acid radical usually taken from sulfuric or hydrochloric acid
Basic dyes
an example of a basic nuclear stain; may be used both as an indicator and as a dye
Methylene blue
Formed by combining aqueous solution of acid and basic dyes
Natural dyes
Capable of staining cytoplasm and nucleus simultaneously and differentially
Natural dyes
examples of natural dyes that are for leukocyte differentiation
Giemsa stain
Leishmann’s stain
One of the principal tissue stains used in histology.
H&E stain
most widely used stain in medical diagnosis and is often the gold standard
H&E stain
H&E stain is the combination of 2 histological stains: [2]
Hematoxylin
Eosin
The hematoxylin stains [cell part] [color] and eosin stains the [cell part] [color], with other structures taking on different shades, hues, and combinations of these
colors.
nuclei
-blue
EM
cytoplasm
-pink
The stain shows the general layout and distribution of cells and provides a general overview of a tissue sample’s structure. Hence a pathologist can easily differentiate
between the nuclear and cytoplasmic parts of a cell.
H&E staining
A natural dye derived from extraction from the heartwood of the mexican tree known as “hematoxylin campechianum”
The tree is grown commercially in ________.
The first person to use hematoxylin in histology
was ________ in [Date]
Hematoxylin
Jamaica
Wadeyerl
1862
Progressive staining unlike in routine type which is a regressive staining because we use _________.
Modified H&E Staining
A type of staining:
➔ No differentiation phase
➔ Used in frozen section
modified h&e staining
May be done by exposing the substance to air and sunlight (slow)
Ripening/Oxidation
Ripening/Oxidation may be done by adding oxidizing agents such as
Hydrogen peroxide
Mercuric oxide
Potassium permanganate
Sodium perborate
Sodium iodate
dangerous if dry
picric acid
Types of hematoxylin [4]
Alum hematoxylin
Iron Hematoxylin
Tungsten Hematoxylin
Copper Hematoxylin
Used in routine H and E
Alum hematoxylin
Mordant in alum hematoxylin
Potash alum/ Potassium aluminum sulfate
_____________
produce good nuclear stain [color]
Alum Hematoxylin
red
Dye examples that are slowly ripened
Erlich’s
Delafield’s
Dye examples that are w/ oxidizing agent: sodium iodate
Mayer’s
Gill’s
Dye examples that are w/ oxidizing agent: mercuric oxide
harris
In Iron Hematoxylin,
________are used as oxidizing agents and mordant
Iron salts
Examples under Iron Hematoxylin
Weigert’s
Heidenhain’s Susa
a type of iron hematoxylin stain that is a ferric chloride
Weigert’s
Weigert is in combination with _________ stain, can
demonstrate_______ elements and
___________ sections
Van gieson’s
CT
Entamoeba histolytica
a type of iron hematoxylin stain that is for muscles/CT fibers
Weigert’s
Standard iron hematoxylin
Weigert’s
Van gieson’s stain is good for demonstrating ______.
collagen
a type of iron hematoxylin stain that is a ferric ammonium sulfate
Heidenhain’s susa
a type of iron hematoxylin stain that is for muscle striations, mitochondira, myelin, chromatin
Heidenhain’s susa
a Mallory’s PTAH/Phospotungistic Acid hematoxylin
Tungsten hematoxylin
For staining muscle striations alone
Tungsten hematoxylin
In tungsten hematoxylin,
To ripen: stand in the _____ for several weeks or use ______ for immediate ripening
light
potassium
Used for the study of spermatogenesis
Copper hematoxylin
H&E staining steps elaborate
- Xylol REHYDRATE [2 changes: descending grade OH]
- Ammonia water submerge [Scott’s tap water]
- Harris/Erlich’s stain
- Tap water rinse
- 1% acid alcohol differentiator [1-2 dips]
- Ammonia water dip
- Tap water rinse
- Submerge OH
- Submerge Eosin
- Tap water rinse
- OH DEHYDRATE [4 changes: ascending grade 95,95,100, 100]
- Xylene- Dealcoholization-Clearing
- mount, label
Removal of pigments is done after _______ and right before ______
rehydration
primary staining
___ -1 stain [nuclear]
___- 2 counterstain [cytoplasmic]
Hematoxylin
Eosin
bridging mordant and dye that will
intensify the color of the stain
Blueing step
Reagents commonly used in staining procedure
- Acid alcohol [differentiator]
- Ammonia water [bluing]
- OG-6 [stains cytoplasm of mature superficial cells]
- Eosin azure [intermediate, para-basal, immature cells]
color bearers _____
auxochromes _____
chromophores
auxochromes
When attracted to the dye molecule, they serve to intensify the color of the dye. They do this by acting as electron donors to the chromophore
auxochromes
the resultant complex of stain-mordant-tissue
Lake
Papanicolau’s stain was developed by [who] [when]
George Papanicolau [1942]
George Papanicolau published 3 formulations of this stain [3 dates]
1942, 1954, 1960
Pap’s smear uses ____________staining
technique
multichromatic cytological
One of the most widely used stains in
cytology, where it is used to aid pathologists in making a diagnosis
Pap’s smear
Although most notable for its use in the detection of ________in the Pap test or Pap smear
cervical cancer
If fluid cytology/ slides – ______
If cell block- ______
Pap’s smear
Routine H&E
Pap’s smear is also used to stain _______
specimen preparations from a variety of bodily ______ and from small needle _______ and organs and tissues
non-gynecological spx prep
fluids
biopsies
Pap smear stains [3]
Hematoxylin
Orange G-6
Eosin Azure- 50
Pap smear procedure
ELEMENTS:
Fix: 95% alcohol for 1 min. Wash: water
Dip: Xylol for 5-10-30 mins
Stains:
Harris hematoxylin: 20-30 mins
OG-6: 3-5 mins
Eosin Azure-50: 3-5mins
PROCEDURE:
1-2: Fix-wash
3-5: Fix- Stain [Harris 20-30 mins]- Wash
6-8: Fix- Stain [OG-6 3-5mins]- Wash
9-11: Fix- Stain [Eosin-50 3-5mins]
12-14: Fix- Xylol dip-Mount/Label
The classic form of the Pap’s stain involves 5 stains in 3 solutions
Generalized Staining Method
In Generalized Staining Method, George Papanicolaou uses ________ in all 3 formulations of the stains he published
Harris hematoxylin
Enumerate 3 formulations in the Generalized staining method
1st:
Hematoxylin -stains nuclei
2nd:
OG-6 + 95% ethyl alcohol + small amount of phosphotungstic acid
3rd:
Bismarck Brown Y + 95% ethyl alcohol + small amount of phosphotungstic acid + lithium carbonate
Eosin Y
Light Green SF Yellowish
Other Formulations Include:
EA-36
EA-50
EA-65
The counterstain is dissolved_______ which prevents cells from over staining which would obscure [2] especially in the case when cells are overlapping on the slide.
95% ethyl alcohol
nuclear detail
cell outlines
added to adjust the PH of counterstain and helps to optimize the color intensity
Phosphotungistic acid
The EA counterstain contains [2], which when in combination, cause both to precipitate out of solution, reducing the useful life of the mixture
bismarck brown
phosphotungstic acid
➔ The stain should result in cells that are fairly _______ so even thicker specimens with overlapping cells can be interpreted
➔ Cell nuclei should be ______, ______in color and the ________ of the nucleus should be well defined.
➔ Cell cytoplasm stains _______ and keratin stains ______ in color
transparent
crisp
blue-black
chromatin patterns
blue-green
orange
Stains the superficial epithelial squamous cells, nucleoli, cilia, and RBC
Eosin Y
Stains the cytoplasm of other cells, other superficial squamous cells
Light Green SF Yellowish
In Light Green SF Yellowish, superficial cells stains _______
In Light Green SF Yellowish, intermediate & parabasal cells _______
orange- pink
turquoise green-blue
Used for staining hemoglobin
Benzidine
has affinity to DNA/RNA
has green fluorescence _______
has red fluorescence ________
Acridine Orange
DNA
RNA
Crystal Violet
For _____ in frozen sections and ______ in blood
amyloid
platelets
Formed by the mixture of crystal violet, methyl violet, and dextrin
Gentian violet
Stained for axis cylinders in embryos
Congo Red
Congo Red is used as a 4% aqueous solution in _____ methods for staining [2]
Krajan
elastic tissue
myelin
Probably the oldest of all stains
iodine
Iodine– stains for [5], and a widely used for removal of [1]
amyloid
carotene
cellulose
starch
glycogen
mercuric fixative pigments
Contrast stain for staining Ascaris eggs and erythrocytes, bacterial spore stain
Malachite green
Demonstrate mitochondria during intravital stain
Janus Green
substitute for carbol fuchsin in acid fast staning
Night blue
Demonstrate neuroglia in frozen section
Victoria Blue
Not real dyes, no auxochromes groups, give color to lipids simply because they are more soluble in lipid medium of the tissues than in their medium of ___% alcohol
Lysochromes [Oil-soluble dyes]
70%
Examples of Oil-Soluble Dyes Used for Demonstration of Intracellular Fats [+color]
Sudan Black B- black
Sudan III- Orange
Sudan IV [Scharlach R]- Red
