SEM100 - MTAP100 - MLSCI 100 | MEDICAL LABORATORY SCIENCE INTERNSHIP

MOUNTING AND STAINING

Lorraine Mission, RMT and Neil John Maravilla, RMT August 18 & September 1, 2021

OUTLINE ● It is somewhat less favored than regressive due to:

I. Staining III. Mounting and Adhesives producing insufficiently intense staining and
II. Stains and Staining INDEX: APPENDIX diffused/obscured details
Solutions ● other parts of the cell will be stained lightly; not specific
Regressive Staining
I. STAINING
● The tissue is first overstained and decolorized
● 9th step of tissue processing (decalcification included)
● Process of applying dyes on the section to see and study Differentiation (Decolorization)
the architectural pattern of the tissue and physical ● Selective removal of excess stain, during regressive
characteristics of the cells staining
● Basic dyes ● In general, if the primary stain used is basic, the
→ has affinity to acidic part differentiation is carried by an acid solution and vice versa
→ e.g. Nucleus stained with hematoxylin → Select the color you want then decolorize the colors
● Acidic dyes you don’t need
→ has affinity to basic part (cytoplasm, etc.) of the cell → basic stain; acid solution for decolorizer
→ e.g. Cytoplasm stained with eosin → acid stain; basic solution for decolorizer
A. MAJOR CLASSIFICATIONS OF TISSUE STAINING Metachromatic Staining
● Histological Staining ● Most dyes stain tissue orthochromatically
→ Tissue constituents are demonstrated by direct → stain tissue with the same color of the dye
interaction with dye/staining solution/. ● Some are metachromatic
● Histochemical Staining → stains tissue different from the color of he dye
→ Tissue is studied through chemical reactions ● This stain belongs to the Thizine & Triphenylmethane
▪ (e.g. Perl’s Prussian blue for iron/hemoglobin & group
PAS for glycogen) ● All metachromatic dyes are cations or basic
→ In enzyme histochemistry, the active reagent serves as → have affinity to acid parts of the cell
the substrate upon which the enzyme act, the final → Therefore all decolorizers for all metachromatic dyes
opacity or coloration produced from the substrate rather are acid
than the tissue ● Examples: (thizine and triphenylmethane)
● Immunochemical Staining → Toluidine blue
→ Allow phenotypic markers to be detected and → Cresyl blue
demonstrated under microscope, using mono or → Safranin
polyclonal antibodies → Basic fuchsin
→ Azure A, B, C
B. METHODS OF STAINING → Methyl violet/crystal violet
● Direct Staining → Bismarck brown
→ Giving color to the sections by using aqueous or → Methylene blue
alcoholic dye solutions
→ e.g. Methylene Blue, Eosin Counterstaining
● Indirect Staining ● Used to differentiate, to provide contrast and background
→ Action of dye is intensified by adding another agent or ● Cytoplasmic stains: Acid dyes; cytoplasm is basic
mordant which serves as a link or bridge → Eosin Y
→ Mordant + Dye = Lake (tissue-mordant-dye complex is → Eosin B
insoluble) ⇒ process is Laking → Phloxine B
→ Accentuator: accelerates or hastens the speed of → Picric acid
staining reaction by increasing staining power and → Orange B
selectivity → Rose Bengal
→ Light Green
Real Life Application: → Lissamine Green
● Lake refers to tissue-mordant-dye complex ● Nuclear Stains: Basic dyes; nucleus is acidic
C. INDIRECT STAINING METHOD → Neutral Red
● Progressive staining → Safranin O
● Regressive staining → Carmine Hematoxylin
● Differentiation (decolorization) → Celestine Blue
● Metachromatic staining → Methylene Blue
● Counterstaining → Toluidine Blue
● Metallic impregnation Metallic Impregnation
● Vital staining ● Tissues are demonstrated not by stain but by colorless
● Intravital staining solution of metallic salts (colorless)
● Supravital staining → Fills in cavities
Progressive Staining → Producing black deposits through reduction
● Stained in a definite sequence until desired intensity of ● Different from stains, it doesn’t incorporate with the tissue
color is attained but it forms precipitate within the tissue
● Once the dye is taken up by the tissue, it is NOT → Examples: gold chloride, silver nitrate
washed/decolorized ● Reagent should not be exposed to sunlight to prevent
explosion
● Inactivated by NaCl or HCl before discarding

INTERNSHIP GGGL, AJDM, KMBG, SCC, JAGT BSMLS 2022 1 of 11

Vital Staining Cochineal + alum + picric Picrocarmine
● The selective staining of living cell constituents acid
demonstrating cytoplasmic structures by phagocytosis of Carmine’s + aluminum Best Carmine (for
the dye particle chloride glycogen)
● Nucleus is resistant to vital staining Cochineal + alum + picric Best Carmine (for
→ the permeability of the dye in nucleus signifies cell acid + aluminum chloride glycogen)
death
Orcein
● eg. eosin-nigrosin for sperm
● Vegetable dye; LICHENS
Intravital Staining ● Colorless, but when treated with NH3 and exposed to air,
● Mostly performed by research laboratories it will produce blue/violet color
→ injected at intrajugular vein of pigs ● Used to stain elastic fibers
● Not commonly found in histopathology department (in
PH), usually seen in radiography department B. SYNTHETIC DYES
● Done by injecting the dye into any part of the animal body ● Also known as “coal tar dyes” or “aniline dyes”
(intraperitoneal, intravenous) Definition of Terms
● Examples: ● Chromophores: substances that can produce visible
→ Lithium colors
→ Carmine ● Chromogens: contains chromophores; differ from dyes
→ India Ink since the color they impart in tissue is not permanent
Supravital Staining ● Auxochrome: substance that when combine with
● Stain living cells immediately after removal from the living chromogen may retain its stain permanently
body → Auxochrome + chromogen = dye
● Thin slices of tissue are placed in staining dishes ● Dye modifiers: substances that affect either the color or
● Common dyes used are: properties of a dye
→ Neutral red → From acidic to basic dye, made possible with dye
→ Janus green modifiers
→ Trypan blue → can range to affecting the color and the properties of
→ Nile blue the dye by addition of a substance
→ Thionine ● Lake: the resultant complex of stain-mordant-tissue
→ Toluidine blue → Laking: the process of staining the tissue
→ New methylene blue for nucleus of red cells
Classification of Chromophore
II. STAINS AND STAINING SOLUTION ● Acid Dye
→ where the active coloring substance is found in the acid
A. NATURAL DYES
component and combines with the basic parts of the
● Cochineal dyes cell
● Logwood dyes → E.g. acid fuchsin, picric acid
● Vegetable extracts ● Basic Dye
Hematoxylin → where the active coloring substances is basic and have
● Extracted from the core or heartwood of Mexican tree affinity to the acidic part of the cell
Hematoxylin campechianum → e.g. methylene blue
● Hematein or haematein is the active coloring agent formed ● Neutral Dyes
by the oxidation also known as ripening → Formed by combining aqueous solutions of acid and
● Ripening may be done by exposing the substance to air basic dyes capable of staining both cytoplasm and
and sunlight (SLOW) this is termed as natural ripening nucleus, simultaneously and differentially
● May be done by adding oxidizing agents such as this is → Romanowsly’s stain, Irishman’s stain, Giemsa stains
termed as artificial ripening C. COMMONLY USED STAINS
→ hydrogen peroxide
→ mercuric oxide (ripening agent of Harris Hematoxylin) Hematoxylin
→ potassium permanganate ● Most commonly used for routine histologic studies
→ sodium perborate ● Commonly used mordants are alum and iron-forming
→ sodium iodate (ripening agent of Ehrlich’s lakes
Hematoxylin) ● Aluminum salts are usually colored blue
Mnemonics ● Ferric salts are colored blue-black
● Aluminum Hematoxylin Solutions used for progressive,
● Si Harris naa sa Mercury kay walay Iodine si Ehrlich
can be used as regressive but requires ‘blueing’
→ GGGL, 2021
→ blueing refers to the process of intensifying the color of
● Mr. Harris and Sir Ehrlich → Maravilla, 2021
the primary stain
Cochineal Dye → Ehrlich’s Hematoxylin
● Old histologic dye extracted from Female Cochineal bug, ▪ hematoxylin is dissolved with alcohol
Coccus cacti ▪ ripened with Na Iodate
● The cochineal dyes when combined with alum (potassium ▪ best for decalcified specimens and frozen sections
aluminium sulfate) it will produce carmine’s dye → Harris Hematoxylin
● Carmine’s dye is used as a powerful chromatin/nuclear ▪ Most preferred because it produces brighter colors
dye ▪ Ripened with Mercuric Oxide
▪ used for routine nuclear staining and sex
Combination Derivative chromosomes
Cochineal dye + potassium Carmine’s dye ▪ stable for long time
aluminum → Cole’s Hematoxylin
Carmine’s + picric acid Picrocarmine ▪ contains celestine blue
▪ routine, alternate for hematoxylin, used for IHC

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 → Mayer’s Hematoxylin ● Malachite green
▪ chloral hydrate is added as a preservative → ascaris eggs, bacterial spore, erythrocytes
▪ Used for glycogen and mucopolysaccharides ● Neutral Red
● Iron Hematoxylin Solutions → Cell granules and vacuoles
→ Weigert’s Hematoxylin ● Night Blue
▪ with ferric chloride → substitute for carbol-fuchsin in AFB
▪ demonstrates muscle fibers and connective tissues ● Orcein
→ Heidenhain’s Hematoxylin → Elastic fibers
▪ Ripened with Ferric ammonium sulfate ● Osmium Tetroxide
▪ Black/dark-grey stains → fat
▪ Good for demonstration of cytoplasmic inclusion, ● Picric acid
muscle striations, myelin → decalcifying agent
→ Phosphotungstic acid Hematoxylin → counterstains in Van Gieson’s
▪ reddish-brown to purple solution ● Prussian Blue
▪ used to demonstrate collagen, nuclei muscle fibers → Colored salt of Ferric Ferrocyanide/iron-staining
Eosin ● Rhodamine B
→ blood/glandular tissue
● Used as a counterstain for hematoxylin, stains cytoplasm ● Toluidine blue
and connective tissues; is the secondary stain → substitute for thionin, NISSL bodies, chromophilic
● It is an acid available for 3 forms: bodies.
→ Eosin Y ● Victoria Blue
▪ Most commonly used; showed green-yellow → Neuroglia in frozen section
fluorescence
→ Eosin B III. MOUNTING AND ADHESIVES
▪ with deeper red color (bluish, erythrosin B) ● 10th step of tissue processing (decalcification included)
→ Eosin S
A. ADHESIVES
▪ Eosin alcohol soluble
● Not necessary for routine staining, but slides will be
Other Stains exposed into varying pH, then it was routinely used
● Acid Fuchsin - Picric Acid ● Optional, but should be applied onto slide before
→ Also known as Van Gieson’s stain used for connective sectioning (to prevent washing out of tissue sample)
tissue and collagen ● Mayer’s Egg Albumin
→ Decalcification in VG stain reaches 37C, collagen is → contains thymol (prevents growth of molds), egg white,
affected in such temperature. and glycerin
● Acridine Orange → formula:
→ differentiates dead and living cells ▪ 50 cc egg white
→ DNA: green ▪ 50 cc glycerin
→ RNA: red ● Plasma
● Acridine Red 3B → most common adhesive; 1 drop of plasma used
→ for calcium and phosphatases enzyme activity → does not affect the results in IHC
● Alcian Blue ▪ recall that we do not use adhesives in IHC
→ a compound similar to chlorophyll specific for mucin → pooled plasma has better adhesion properties than
and connective tissues non-pooled plasma
● Aniline Blue ● Dried albumin
→ use as counterstain → dried albumin, NaCl, dissolved in 100cc of distilled
→ stains cytoplasm water
● Basic Fuchsin ● Gelatin 1%
→ plasma stain ● Gelatin-formaldehyde mixture
→ use to stain acid-fast microorganism, mitochondria, and ● Starch paste
smooth muscles ● Poly-L-Lysine
● Benzidine ● APES (3-aminopropyltriethoxysilane)
→ Hemoglobin
B. MOUNTANTS
● Bismarck Brown
→ use to stain diphtheria ● Usually syrupy fluid, preserves slide and for easy handling
● Carmine and storage, prevents distortion during microscopic exam
→ Chromatin ● Should give good refractive index
● Celestine blue ● Slides must be wiped with xylene before mounting
→ used to stain nucleus/chromatin ● Once mounted, it can be incubated at 37oC for 24 hours
● Congo red ● Good mountant should have a refractive index of 1.518
→ stains the axis cylinders of embryos, elastic fibers → 1.518 refractive index of glass
amyloid, myelin Characteristics of a Good Mountant
● Crystal Violet ● Avoid distortion of the image, refractive index of the
→ Nuclear, Chromatin mountant should be as near as possible to that of the
● Giemsa class which is 1.518
→ blood, differentiates leukocytes ● Should be freely miscible with xylene and toluene
● Gold Substitute ● Should not dry quickly
→ Metallic Impregnation ● Should not crack or produce artifactual granularity on the
● Iodine slide upon drying
→ oldest stain ● Should not dissolve or fade out tissue sections
→ starch, glycogen amyloid ● Should not cause shrinkage and distortion of tissues
→ removal of mercury ● Should not leach out any stain or affect staining
● Janus Green B ● Should not change color or pH
→ Mitochondria ● Should set hard

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 C. TYPES OF MOUNTING MEDIA H and E Staining: Real Life Application
● For routine diagnosis, the use of H&E is by far preferred
Aqueous Mounting medium
for viewing cellular and tissue structure detail by
● Usually for lipids because resinous media contain xylene pathologist
which may dissolve fats ● The staining procedure for H and E
● Water → Dewaxing
● Glycerin - RI 1.46 ▪ 2-3 changes of xylene, done in the oven
● Farrant’s Medium - RI 1.43 → Rehydration
● Apathy’s medium - RI 1.52 ▪ descending grades of alcohol
● Brun’s Fluid: recommended for mounting frozen sections → Hematoxylin
from water → Differentiation
Resinous Mounting medium ▪ 0.3% acid
● Used for prep that have been dehydrated and cleared → Bluing
● Canada balsam ▪ Can be ammonia water
→ natural resin extracted from Canadian tree, Abus ▪ Intensifies the effect of the primary stain
balsamea, with RI of 1.524 → Eosin
● synthetic resins used for embedding undecalcified bones, ▪ Some laboratories soak in 95% ethanol before
for EM staining in Eosin
● DPX - RI 1.532 → Dehydrating
● XAM - RI 1.52 ▪ Ascending grades of alcohol in two changes
● Clarite - RI 1.544 → Clearing
▪ Clear again in two changes of xylene
Mounting Media RI Other details → Cover-slipping
AQUEOUS MOUNTING MEDIA
water low temporary mounting
glycerin 1.46 semi-permanent
glycerin jelly 1.47 standard mountant
for fat stains
Farrant’s medium 1.43 standard mountant
for fat stains
Apathy’s medium 1.52 used for methylene
blue-stained nerve
preparations
does not require ringing
Brun’s fluid recommended for
mounting frozen sections
from water
RESINOUS MOUNTING MEDIA
Canada balsam 1.524 oleoresin
darkens slightly with age
recommended for whole
mounts and thick sections
● Wash in every step except the transition from xylene to
DPX 1.532 recommended for small alcohol and eosin to alcohol steps
tissue sections but not ● 95% ethanol step is optional; may proceed directly to
whole mounts eosin
used in excess amounts
XAM 1.52 resin mixture in xylene Troubleshooting
Clarite 1.544 generally preferred over ● Most of the time, troubleshooting procedures include
DPX increasing/decreasing the time of exposure to
Other recommended synthetic mounting media: hematoxylin/eosin
● Permount → made by Fischer Scientific
● HSR → Harleco Synthetic Resin
● Clearmount → Gurr
Ringing
● Sealing of margin of the coverslip.
● Use to prevent escape of fluid or semi-fluid samples
● Evaporation of mountant
● To immobilize the coverslip
● To prevent sticking of the slides upon storage
● Examples:
→ Kronig’s Cement:
▪ paraffin + powdered colophonium resin
→ Durofix
;

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 INDEX: APPENDIX

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● Stains for Electron Microscopy
→ Phosphotungstic Acid
→ Uranyl acetate
→ Lead

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