MODULE 5 - Staining & Mounting Flashcards
Process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells
staining
Tissue components are demonstrated by direct interaction with a dye or staining solution
Histological stain
used for chemical component/reaction
Histochemical stain
Active tissue component is colored
Histological stain
Examples of histological stains
Micro-anatomical stains
Bacterial stains
Specific tissue stains
Uses antibodies instead of stains
Immunohistochemical stain
Perl’s Prussian blue reaction is for
hemoglobin
Periodic acid Schiff is for
Carbohydrates
Examples of histochemical stains
Perl’s prussian blue
Periodic and schiff
Detection of phenotypic markers that are detected by antibodies
immunohistochemical stain
Examples of immunohistochemical staining
monoclonal
polyclonal
uses aqueous or alcoholic dye solutions to produce a color
Direct staining
Uses only one dye, usual color is the resulting color
direct staining
Example of direct staining
methylene blue
Uses mordant/accentuator
Indirect staining
Link/bridge between dye and tissue
mordant
Examples of mordant and solutions that contains them
Potassium alum- Meyer’s hematoxylin
Iron (Ferric ammonium chloride)- Weigert’s hematoxylin
What is accentuator
It heightens the color intensity and selectivity
Increases staining power
Examples of accentuator and solutions that contains them
KOH- Loeffler’s methylene blue
Phenol- carbol fuschin or carbol thionine
tissue elements re stained in a definite sequence
gradual application of dyes
No excess dye No decolorization
Progressive staining
Tissue is overstained
Regressive staining
Excess dye in regressive staining are removed by
Decolorization/differentiation
Example of regressive staining
Acid alcohol- can remove both acid and basic dyes
staining tissue with a color that is different from the stain itself
Metachromatic staining
Examples of metachromatic staining
Methyl violet or crystal violet Cresyl blue for reticulocytes Safranin Bismarck brown Basic fuschin Methylene blue Thionine Toluidine blue Azure A,B,C
involves application of a different color to produce contrast background
Counterstaining
Example of counterstain and explain its use
Eosin- red acid dye for cytoplasmic stain
Forms of eosin
✓ Eosin Y- yellow
✓ Bluish- eosin b
✓ Ethyl Eosin- eosin s
tissue elements are demonstrated NOT by stains but by colorless solutions of metallic salts
Metallic impregnation
When applied to tissues, it is not absorbed and can only leave black deposits on surface
Metallic impregnation
Examples of metallic impregnation
Ammoniacal silver
Silver nitrate
Gold chloride
Demonstrates living cells, fresh and viable cells.
Vital staining
2 types of vital staining
Intravital
Supravital
Dye is injected to any part of the body
Intravital
Examples of intravital stains
Lithium
Carmine
India ink
stain is applied immediately to tissue after its removal from living body
Supravital
Best vital stain
Neutral red
Supravital stain for mitochondria
Janus green
Examples of supravital stain
Neutral red- best vital stain Janus green- for mitochondria Trypan blue Nile blue Toluidine blue Thionine
Most common method utilized for microanatomical studies of tissues
Rountine H&E
H&E stain is what type of stain
Regressive staining
Steps in H&E staining
- Initial xylene bath- for further deparaffinization
- Descending grades of alcohol- for hydration
- Hematoxylin- primary dye, nuclear stain-basic dye
- Acid alcohol- decolorizer
- Ammonia water- bluing agent
- Eosin- counterstain, cytoplasmic stain-acid dye
- Ascending grades of alcohol- for dehydration
- Final xylene bath- for clearing prior to mounting
Enumerate H&E results
Nuclei- blue to blue black
Karyosome- deep blue
Cytoplasm- pale pink
RBCs, eosinophilic granules, keratin- bright orange red
Calcium and decalcified bones- purplish blue
Decalcified bone matrix, osteoid, collagen- pink
Muscle tissue- deep pink
Staining Methods for FROZEN SECTION
H&E
Thionine
Polychrome methylene blue
Alcohol pinacyanol method
– derived from plants and animals
natural dyes
Examples of natural dyes
Hematoxylin
Cochineal dyes
Orcein
Saffron
Hematoxylin is a stain
True or False
False
active coloring agent of hematoxylin
Hematane
Hematoxylin is formed from the oxidation of hematoxylin either by
ripening (Exposure to sunlight)
adding oxidizing agents
Oxidizing agents in hematoxylin
Hydrogen peroxide Mercuric peroxide Sodium iodate Sodium perborate Potassium permanganate
Hematoxylin is used in combination with
alum
iron
chromium
copper salts
ALUM HEMATOXYLIN uses _______ as mordant
potassium alum
Ripening agent of Ehrlich’s hematoxylin
Sodium iodate
Ripening agent of Harris hematoxylin
Mercuric oxide- for exfoliative cytology and sec chromosomes
Ripening agent of Cole’s hematoxylin
Alcoholic iodine
Ripening agent of Mayer’s hematoxylin
Sodium iodate
Contains citric acid and chloral hydrate as preservative
preferred for immunohistochem
Ripening agent of Delafield’s hematoxylin
Glycerol- to stabilize oxidation process
Smells like wine, color purple-red shade
Ripening agent of Gill’s hematoxylin
sodium iodate
used to stain mucin in goblet’s cell
Ripening agent of Carazzi’s hematoxylin
Potassium iodate
– in general for photomicrography
IRON HEMATOXYLIN
Weigert’s Hematoxylin is for?
and what is its component/mordant
Muscle fibers and CT
Ferric ammonium chloride- both mordant and ripening agent
Heidenhain’s Hematoxylin is for?
and what is its component/mordant
nucleus and cytoplasm (mitochondria)
Ferric ammonium sulfate- both mordant and ripening agent
COPPER HEMATOXYLIN is used for
spermatogenesis
TUNGSTEN HEMATOXYLIN contains
phosphotungstic acid
LEAD HEMATOXYLIN is for
demo of granules of endocrine cells of alimentary tract
MOLYBDENUM HEMATOXYLIN is for
demo of collagen, reticulin and argentaffin cells
extracted from bug treated with alum to produce carmine
Cochineal dyes
Cochineal dyes is treated with alum to produce
carmine
Cochineal dyes with picric acid will produce
picro carmine- for neuropathological studies
Cochineal dyes with aluminum chloride will produce
Bescarmine- glycogen
– vegetable dye extracted from lichens and is used for ______
Orcein
staining elastic fibers
vegetable dye extracted from lichens to be used as pH indicator because of poor staining property
Litmus
Synthetic dyes also known as
Coar tal/aniline dyes
2 components of Synthetic dyes
- Chromophore- coloring property
2. Auxochrome- dyeing propert, to retain the imparted color
what is lysochrome dyes
also known as Oil soluble dyes, used for FATS
It has no auxochrome
Most sensitive soluble dye
Sudan black B
Sudan IV also known as and what is its use
Sharlach R
Neutral lipids staining them red
First sudan dye to be introduced in histochemistry
Sudan III
Sudan III is good for
fat stain for CNS tissues
Sudan Black B, Sudan IV and Sudan III are considered what type of stain
Histochemical stain
Other examples of histological stains
Wrights Giemsa Methylene blue Crystal violet Carbol fuschin
Accentuator is a part of staining reaction
True or False
False
Components of acid alcohol
HCL + ethanol
What is orthochromatic staining
Tissue stained with same shade/hue as the dye
– uses aqueous or alcoholic dye solutions to produce a color
Direct Staining
Metachromatic staining is used to stain what structures
Cartilage
Amyloid
Mast cell granules
Epithelial mucins
2 examples of staining jar
couplin jar
slotted staining dishes
Stains under Alum hematoxylin
Gills
Ehrlichs
Meyers
Coles
Process of applying a mounting medium between the stained tissue section and the coverslip
mounting
Reasons for doing mounting
permanent safekeeping
easy handling and storage
prevent bleaching of sections
Mounting medium should have a refractive index close to that of the slide which is
1.518
What is resinous mountant
dehydrated and cleared by xylene
permanent
What is aqueous mountant
for water miscible preparation
semi-permanent, temporary
Canada balsam’s refractive index is
1.524
Routine mountant
Canada balsam
Canada balsam is extracted from
Abus Balsamea
Disadvantage of using canada balsam
Darkens with age
causes gradual fading of stains
Enumerate types of resinous mountant and their refractive index
Canada balsam- 1.524
DPX- 1.532
XAM- 1.52
CLARITE- .544
Glycerin jelly’s aka ______ and refractive index
Kaiser’s
1.47
Farrant’s aka ______ and refractive index
Gum arabic
1.43
Apathy’s refractive index
1.52
for methylene blue stained nerve preparations
Brun’s is used for
frozen sections directly from water
Water as mountant
not used because of its low refractive index
easily evaporates and cannot examine those under IOI
Ringing is mandatory
True or False
False
Sealing margins of coverslip
ringing
Ringing is done to
prevent escape of fluid
prevent evaporation
prevent sticking of slides upon preparation
immobilize coverslip
Type of ringing medium
Kronig cement
durofix
Color that eosin stains
pink
color that hematoxylin stains
blue
examples of NEUTRAL / AMPHOTERIC DYE
Wrights
Irishman
Giemsa
Leishmann
Process of coating slide with thin celloidin solutions. Done if sections will be subjected to strong alkali or acid solutions and for tissues containing glycogen for demonstration
COLLODIONIZATION
Solvents Used for Stains
Water
Alcohol (methyl or ethyl)
Aniline water
Phenol
Steps in RE-STAINING of OLD SECTIONS
- immerse in xylene for 24 hrs
- remover coverslip
- immerse in xylene for 30 mins
- immerse in 0.5% potassium permanganate for 5-10 mins. rinse with tapwater
- immerse in 5% oxalic acid
- re-stain
Years of retention for Clinical pathology laboratory reports
10 yrs
Years of retention for Autopsy forensic reports
indefinitely
Years of retention for Surgical pathology and bone marrow Reports
10 yrs
Years of retention for Cytogenetics report
20 yrs
Years of retention for Pathology /Bone marrow slides
10 yrs
Years of retention for Pathology blocks
10 yrs
Years of retention for Cytogenetic slides
3 yrs
Years of retention for Cytogenetic diagnostic images
20 yrs
it governs the contrast between the cellular detail and the background, and also the transparency of the observed sample against the bright field of the microscope.
refractive index
The mounting media must always have an RI _____ than the mounted sample to impart more transparency.
higher
The slide may then be incubated at __________ after mounting, to harden the medium.
37 degC
12-24 hrs
You can use immersion oil on an uncovered slide
True of False
False
Oil can be applied, but do not attempt to wipe it off until the mounting medium is cured for how long?
at least overnight or in a hot plate or oven for 1 hr
Do not store mounted slides vertically for ______ if cured at room temperature.
2 days
Excessive mounting medium will cause it to ooze out of the sides of the cover glass, and should be carefully wiped with a fine cloth moistened with ____________.
xylene
Excessive blotting will
dry up the section, causing shrinkage and cracking of the specimen.
If the section has to be remounted, the cover glass may be removed by soaking in
xylene
Excess xylene, if not removed, will
mix with the mountant and form bubbles on the slide.
Too little mounting medium may
What is the remedy
cause improper setting of the coverslip or formation of bubbles on the section
teased out by gently pressing on the cover glass with a pointed forceps or mounting needle.
Setting may be hastened in
hot oven at 50°C for 2 hours.
basic aniline dyes should be mounted in
non-acid containing mountants
Prussian blue reaction should be mounted in
non-reducing media
a paper label should contain
patient’s name
section number
staining method used
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 MEDIA
glycerin jelly or gum arabic used to
solidify the medium
glycerol used to
cracking and drying of the preparation
sugar is used to
increase the refractive-index
usually regarded as the standard mountant for fat stains.
glycerin
standard mounting medium used when dehydration and clearing with xylene cannot be made
glycerin jelly
recommended as an alternative for glycerine jelly.
Polyvinyl alcohol
has the highest index of refraction
Pure glycerin
used as a mountant in immunofluorescence microscopy
Polyvinyl alcohol
The mountant is not set in the desired amount of hardness and therefore requires “ringing”
glycerin jelly
does not solidify upon storage and therefore does not need to be heated before use.
gum arabic medium
general purpose aqueous mountant
apathy’s
not compatible with normal histological stains.
von apathy’s
It is one of the most useful aqueous mountants for fluorescent microscopy, being virtually nonfluorescent.
apathy’s
Canada balsam can be made neutral or acid by adding excess amounts of
calcium carbonate
salicylic acid
It is a transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation
Canada balsam
substitute for xylene in canada balsam that may be less ofa health hazard but may cause other problems such as slow hardening and premature darkening.
histomount
may be substituted for xylene as a solvent in canada balsam. The medium can only be neutralized temporarily since the mixture becomes acidic and changes into a brown color upon storage.
Benzene
may be added to canada balsam to maintain its neutral reaction
calcium carbonate chips
Canada balsam is recommended for
whole mounts and thick sections
This is a resinous medium recommended for small tissue sections but not for whole mounts because of shrinkage produced on drying
DPX
It has a greater advantage over Canada balsam in that slides can be cleaned of excess mountant simply by stripping it off after cutting around the edge of coverslip
DPX
generally preferred over D.P.X
Clarite
generally suitable for all enzymatic label/chromogen combinations and fluorescent labels.
Aqueous mounting medium
quenches the staining intensity.
glycerol
exposure to excitation light of most fluorescent labels which results in diminished staining
photo bleaching
may form when the mounting media is too thin, and as it dries air is sucked in under the coverslip.
bubbles
Contamination of clearing agents or cover slipping media may
produce a bubbled appearance under the microscope.
