Special Stains Flashcards
Define special stains
Staining methods that use dyes, metal impregnation, or chemical reactions to demonstrate tissue structures.
Trichrome stains
- differentiates CONNECTIVE TISSUE from other elements
- uses three ACIDIC dyes*
- based on porosity: smallest dye stains all tissue elements; larger dyes “evict” smaller molecules in larger pores
*NOTE: alum hematoxylin is unsuitable because it is differentiated in acidic pH
Which hematoxylin is used in Trichrome stains ? Why ?
Weigert’s (iron) hematoxylin; alum hematoxylin is differentiated by acidic pH
Masson Trichrome
- differentiates between SMOOTH MUSCLE vs COLLAGEN in tumors
- demonstrates increased collagen in cirrhosis
Principle: Masson Trichrome
- Bouin’s fluid at 50°C for 1 hour
- Biebrich scarlet + Acid fuchsin = STAINS all ACIDOPHILIC elements RED
- Phosphomolybdic/tungstic = colorless dye “evicts” red stain from larger pores (ie. collagen)
- Alanine Blue = largest dye stains collagen BLUE
Color: Nuclei in Masson Trichrome
grey to black
Color: Cytoplasm, keratin, muscle fibre, erythrocytes in Masson Trichrome
Red
Color: Collagen in Masson Trichrome
Blue
Cause+Troubleshoot: Masson Trichrome stain all decreased
Cause: failure to pre-mordant with Bouin’s when using formalin-fixed tissue
Troubleshoot: pre-mordant for 1 hour at 60°C
What can be used in place of analine blue for collagen staining in Masson Trichrome ?
Light green, SF yellowish
T or F: In Masson Trichrome, the nuclear stain does not have to be very dark or demonstrate fine chromatin patterns
TRUE; the nuclear stain is for contrast and orientation; it does not have to be very dark or demonstrate fine chromatin patterns
Why should little spills of picric acid from Bouin’s fluid be wiped away immediately ?
Dry picric acid is explosive
2 stains to demonstrate elastin
- Verhoeff’s
- Gomori’s aldehyde fuchsin
Elastin fibres contain __ bridges, which oxidizes to form __ derivatives. These substances are strongly (acidophilic/ basophilic).
Elastin fibres contain DISULFIDE bridges, which oxidizes to form SULFONIC ACID derivatives. These substances are strongly BASOPHILIC.
Principle: Verhoeff Van Gieson
- tissue is OVER-STAINED with IODINE/ FERRIC CHLORIDE/ HEMATOXYLIN
Ferric Chloride = mordant + oxidizer
Iodine = oxidizes hematoxylin and elastin - differentiate using EXCESS FERRIC CHLORIDE = excess mordant breaks tissue-mordant complex*
- iodine is removed by sodium thiosulfate (aka HYPO)
- Van Gieson = counterstain
*NOTE: elastin has the highest affinity for verhoeff and would be decolorized last
Color: Elastin in Verhoeff Van Gieson
Black
Color: Collagen in Verhoeff Van Gieson
Red
Color: Muscle in Verhoeff Van Gieson
Yellow
In Verhoeff Van Gieson, the collagen fibres should remain __ during differentiation to avoid removal of stain from elastin fibres
In Verhoeff Van Gieson, the collagen fibres should remain PALE GREY during differentiation to avoid removal of stain from elastin fibres
What must be done after differentiating with Ferric Chloride in Verhoeff Van Gieson staining ?
- Rinse in distilled water to stop differentiation
- Slides must be checked microscopically (collagen must remain pale grey)
Gomori’s Aldehyde Fuchsin is described as “empirical.” What does this mean ?
- we are unsure of why it stains elastin
- elastin stains with both eosin (anionic) and basic fuchsin (cationic), so it can’t be simply ionic binding
- hydrogen bonding is hypothesized to be a contributor
3 functions of Ferric Chloride in Verhoeff’s
- Mordant
- Oxidizer
- Differentiator
Verhoeff and Gomori: progressive or regressive ?
Verhoeff = regressive
Gomori’s Aldehyde Fuchsin = progressive
Principle: Gomori’s Aldehyde Fuchsin
- ACIDIFIED PARALDEHYDE + alcoholic BASIC FUCHSIN = RIPEN for 2-4 DAYS before staining
- progressive
- (optional) Potassium permanganate = intensifies staining
Color: Elastin (and beta granules in Pancreas, mast cells, sulfated mucins and cartilage) in Gomori’s Aldehyde Fuchsin
Purple
Color: Potassium permanganate counterstain in Gomori’s Aldehyde Fuchsin
Green
Storage temp of ripened Gomori’s aldehyde fuchsin
4°C
Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with __ if desired.
Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with 70% ALCOHOL if desired.
What kind of stains demonstrate Reticulin ?
Silver stains
Principle: Gordon and Sweet’s Reticulin Stain
- potassium permanganate oxidizes Reticulin = aldehyde groups of reticulin
- oxalic acid bleaching removes discolouration from permanganate
- (INDUCED ARGYROPHILIA) Ferric ammonium sulfate = sensitizer binds to aldehydes; to be replaced by silver from ammoniacal silver solution
- formalin reduces silver to metallic form
- gold chloride tones silver = brown to black
- sodium thiosulfate removes any unreduced silver*
- counterstain: nuclear fast red or eosin
*NOTE: prevents section from darkening when exposed to light
Color: Reticulin in Gordon&Sweet’s Reticulin Stain
Black
Color: Counterstain in Gordon&Sweet’s Reticulin Stain
Red
During Gordon&Sweet’s Reticulin staining, a white precipitate forms on the walls of the jar/ solution surface. What does this mean ?
- glassware was NOT acid washed/ bleached + rinsed with dH2O
- silver solution is CONTAMINATED
- staining will be weak
Why are Reticulin silver stains usually made fresh ?
Ferric AMMONIUM sulfate + SILVER = EXPLOSIVE
- discard after use
- avoid sunlight
How are silver solutions neutralized ?
- add CHLORIDE ions (HCl, bleach, or saline)
- precipitate out the silver = silver chloride
- dispose in HALOGENATED waste
2 stains to demonstrate carbohydrates
- PAS (Periodic Acid Schiff)
- Alcian blue
Which carbohydrates are PAS positive ?
- glycogens
- NEUTRAL mucins (mucopolysaccharides)
NOTE: basement membrane, fungi (C. albicans), zymogen granules, colloid, and reticulin are also POS
Which carbohydrates are Alcian blue positive ?
(ACID mucopolysaccharides):
- carboxylated mucins
- sulfated mucins
Glycogen: PAS vs PAS-D
PAS = POS
PAS-D = neg
Principle: Periodic Acid Schiff
- periodic acid oxidizes hydroxyl in carbs = aldehydes form
- schiff reagent (basic fuchsin + sulfurous acid*) reacts with aldehydes = bind to tissue
- water rinses removes loose sulfurous acid groups = restores qunoid ring = BRIGHT RED
*NOTE: reduction causes quinoid ring to be lost = colorless
Why is periodic acid used to form aldehydes in PAS ?
Periodic acid is a WEAK OXIDIZER
- others will oxidize hydroxyl beyond aldehydes
What is Schiff reagent made of ?
Basic fuchsin + sulfurous acid
Color: PAS-positive tissue elements
Magenta
Cause+Troubleshoot: PAS Non-specific staining
Cause: did not rinse excess Schiff reagent
Troubleshoot: water rinse
In PAS, __ is important for proper color development
In PAS, the WATER RINSE is important for proper color development
PAS-D
Periodic Acid Schiff with Diastase (α-amylase):
- digestion step = specific for glycogen
Principle: PAS-D
- diastase (α-amylase) breaks down glycogen into SMALLER sugars (maltose + glucose)
- water rinse WASHES AWAY SIMPLE SUGARS
- stained in duplicate: PAS-D vs PAS
- INDIRECT DETECTION = when compared to PAS, absent staining on the digested slide (PAS-D) will be due to glycogen
Color: glycogen on PAS-D
UNSTAINED bc of digestion+rinse
Color: PAS-pos tissue elements on PAS-D
Magenta
NOTE: EXCEPT glycogen
Source of α-amylase for PAS-D
Saliva
Alcian blue
- a copper pthalocynanin CATIONIC dye
- stains ACID MUCINS to differentiate adenocarcinomas
Principle: Alcian blue
- at pH 2.5, IONICALLY binds with carboxylated and sulfated (acid) mucins
- at pH 1.0, only sulfated acid mucins will stain
Color: at pH 2.5, carboxylated/ sulfated mucins in Alcian blue
Dark blue
Color: at pH 1.0, sulfated mucins in Alcian blue
Light blue
Color: Nuclei in Alcian blue
Red
T or F: Alcian blue will appear more lightly stained in a lower pH
TRUE; Alcian blue will appear more lightly stained in a lower pH
- sulfated mucins will be light blue in pH 1.0, but darker at pH 2.5
How is a low, stable pH achieved for Alcian blue staining ?
Acid rinse prior to staining
Alcian Blue + PAS
- Stain Alcian blue first = stains acid mucins BLUE
- PAS = stains neutral mucins MAGENTA
- tissue elements that contain both acid and neutral mucins = purple
Amyloid (2 types)
- amorphous, eosinophilic protein deposits associated with amyloidosis
- β-pleated sheet structure of proteins
1. AA amyloid/ secondary= serum “amyloid A” proteins = inflammation and chronic disease
2. AL amyloid/ primary = kappa or lambda immunoglobulins = B-cell disease (ie. MM)
Principle: Congo red
- demonstrates AMYLOID
- congo red fits within β-PLEATED SHEETS of protein, stabilized by HYDROGEN BONDING = red
- attaches to protein at regular intervals = APPLE-GREEN birefringence under polarized light
Color: Amyloid in Congo red
Red; apple green under polarized light
Color: elastin in Congo red
Pale pink
Color: nuclei in Congo red
Purple
Required thickness of tissue to observe polarized APPLE GREEN color (Amyloid) in Congo red
7-10 μm
Hemosiderin
- yellow-brown ENDOGENOUS pigment
- storage form of iron = ferric hydroxide
- normally stored in bone marrow + spleen
- HEMOSIDEROSIS = excess hemosiderin stored in other organs
Principle: Perl’s Prussian Blue
- HISTOCHEMICAL STAIN
- detects FERRIC iron = reacts with acid-ferrocyanide = insoluble BLUE pigment
- ferrous iron will not react
Color: Hemosiderin in Perl’s prussian blue
Prussian blue
Color: Counterstain in Perl’s Prussian blue
Pink
Why must deionized/ distilled water be used in Perl’s Prussian blue staining ?
Tap water can contain iron = non-specific blue precipitate
Why can’t decalcification be done on tissues stained for Perl’s Prussian blue ?
Iron can be dissolved away = false negative
Prinicple: Oil red O
- a Sudan dye; demonstrates fat/ lipids
- uses Oil red O dissolved in isopropyl alcohol
- SELECTIVE SOLUBILITY = Oil red O is more soluble in fat and will MIGRATE FROM SOLVENT TO FAT in tissue
Color: Fat in Oil Red O
Red
Color: Nuclei in Oil red O
Purple-blue
Cause+Troubleshoot: In Oil red O, fat droplets are found throughout tissue
Cause: coverslipped was squished to remove air bubbles
Troubleshoot: coverslip gently and avoid heating slide with fingers
T or F: paraffin embedded tissue can be used to demonstrate fat
FALSE; fat is readily soluble in alcohols and xylene so tissue cannot be processed; FROZEN SECTIONS are needed to demonstrate fat
Fontana Masson
- ARGENTAFFIN stain (binds and reduces silver)
- demonstrates melanin and granules of enterochromaffin cells in small intestine
Principle: Fontana Masson
- diamine silver applied to tissue
- argentaffin substances will bind and reduce silver
- gold chloride to tone tissue
- sodium thiosulfate (hypo) removes any unreduced silver
- counterstain: Nuclear-fast red or Eosin
Color: Melanin and Argentaffin granules in Fontana-Masson
Black
Color: Nuclei in Fontana-Masson
Pink
How can FM be made specific for Melanin ?
- add a duplicate slide: FM + bleach (potassium permanganate OR hydrogen peroxide)
- when compared to the regular slide, absent staining on bleached slide will be due to melanin
T or F: do not use metallic forceps for any silver method
TRUE; using metallic forceps for any silver method can be a source of contamination
2 stains for calcium
- Von Kossa’s Silver stain
- Alizarin Red S
Von Kossa’s Silver stain vs Alizarin Red S
- both used to demonstrate calcium
Von Kossa’s Silver stain:
- INDIRECT; silver ions replace calcium bound to phosphate groups = reduced to visible silver
- calcium is assumed to be where visible silver is detected
Alizarin Red S:
- DIRECT; binds calcium present in tissues
- other metals will react as well, but are insignificant
Principle: Von Kossa’s Silver stain
Metallic substitution:
- INDIRECTLY detects calcium
- silver ions react with phosphate/ carbonate groups of calcium phosphate/ carbonate
- bright light reduces silver to its metallic form
Color: Calcium in Von Kossa’s Silver stain
Black
Color: Background stain in Von Kossa’s Silver stain
Pink
What is generally a cause for non-specific staining ?
Contaminated glassware
Cause+Troubleshoot: In Von Kossa’s Silver stain, calcium appears brown instead of black
Cause: insufficient light to reduce silver to its metallic form
Troubleshoot: extend use of sunlight or UV lamps
Gram pos vs Gram neg bacteria
Gram pos = thicker cell wall of PEPTIDOGLYCAN + lipoproteins
- retain crystal violet during decolorizing = purple
Gram neg = thinner walls with a single layer of peptidoglycan + MORE LIPOPROTEINS
- lipoproteins layers disrupted by alcohol = crystal violet is decolorized
- counterstained pink with safranin
Principle: Modified Gram Stain
- crystal violet stains both gram pos/ neg bacteria
- iodine (mordant)
- alcohol dissolves lipoprotein layer, while leaving peptidoglycan intact
Gram pos: retains crystal-violet-iodine complex due to layers of peptidoglycan
Gram neg: is decolorized
- counterstained
Color: Gram pos bacteria in Modified gram stain
Purple
Color: Gram neg bacteria in Modified gram stain
Pink
Gram stain modified using Brown-Hopps uses what to stain which colors ?
Basic fuchsin = gram neg bacteria = pink
Picric acid = tissue elements = yellow
Gram stain modified using Gram-Twort uses what to stain which colors ?
Neutral red = gram negative bacteria = red
Light/ fast green FCF in alcohol = collagen + cytoplasm = green
Ziehl-Neelsten Stain
demonstrates acid-fast organisms (ie. Mycobacterium) = have thicker cell walls with WAXY MYCOLIC ACIDS
Principle: Ziehl-Neelsten stain
- alcoholic phenol helps basic fuchsin to PENETRATE cell wall = IONICALLY binds acid-fast bacteria = PURPLE
- dilute acid solutions; true acid-fast bacteria will RESIST acid decolorization (waxy mycolic acids on cell wall)
Color: Acid-fast bacilli in Ziehl-Neelsten stain
Purple
Color: Background stain in Ziehl-Neelsten stain
Methylene Blue
Hazard of alcoholic phenol
Highly toxic by absorption and inhalation
Diff-Quik
Giemsa stain used to screen for Helicobacter pylori:
- cationic dye = methylene blue
- anionic dye = eosin
NOTE: any bacteria will stain blue, looking at morphology is important
Principle: Diff-Quik
- Eosin stains positively-charged tissue elements (Cytoplasm)
- Methylene blue stains negatively-charged tissue elements (Bacteria, nuclei)
What kind of stains are used to demonstrate Spirochetes ?
- silver stains; spirochetes are argyrophilic (bind silver)
- but NEEDS an EXTERNAL REDUCING AGENT to visualize the results (ie. hydroquinone in Warthin-Starry)
Principle: Grocott’s Methenamine Silver stain
- chromic acid oxidizes FUNGAL cell walls = FORMS ALDEHYDES
- in warm methenamine-silver solution, silver binds to aldehydes = reduced to metallic silver (INDUCED ARGENTAFFIN)
- gold chloride; tones silver
- sodium thiosulfate; removes unreduced silver
- counterstain: Light green
Color: Fungus in Grocott’s methenamine silver
Black
Color: Mucin in Grocott’s methenamine silver
Grey
Color: Counterstain in Grocott’s methenamine silver
Light green
You find out your aldehyde fuchsin has expired when you are ready to perform Gomori’s aldehyde fuchsin stain. Why is this an issue ?
It takes 72 hours for the solution to ripen; patient results will be delayed 3 days
Purpose of toning in silver stains
some silver is replaced by gold ions = transforms color from dark brown to black
Why must silver solutions be neutralized prior to disposal ?
Silver solutions (especially when containing ammonia) can be explosive when exposed to metal pipes
- also very toxic
Why do tissue sections tend to fall off slides during silver staining ? How can this be prevented ?
Due to the alkaline nature of silver stains, using charged slides is recommended
Best overall control for PAS stain. Why ?
Kidney = basement membrane contains small amounts of aldehydes after oxidation, making the control more sensitive
How is glycogen thought to be fixed ? Why should tissue be fixed immediately ?
- Glycogen is not affected by formalin; it is trapped within the fixed protein of the cell
- Glycogen breaks down into glucose* quickly; tissue must be fixed immediately to trap glycogen
NOTE: since glucose is highly soluble, it will dissolve in the fixative = false neg
Why is glutaraldehyde not an acceptable fixative for PAS staining ?
- Schiff reagent will stain any aldehydes present in tissue (which we create by oxidizing polysaccharides)
- glutaraldehyde will leave free aldehydes in tissue = binds to Schiff reagent = false pos
Periodic Acid Schiff: How can you determine if there are pre-existing aldehydes in your tissue ?
Skip the oxidation step; any positivity with Schiff reagent = pre-existing aldehydes
Why are slides rinsed in dye solvents before staining with Alcian blue ?
- Alcian blue is pH-dependent
- an acid rinse prior to staining ensures stable pH = demonstrates substances (carboxylated/ sulfated mucins) we intend to
Why is it important to keep the Counterstain light ?
- avoid masking positive results
- counterstain only provides a morphological overview
Why should tissue for amyloid controls be processed and embedded instead of stored in formalin until required ?
Amyloid staining becomes weaker with extended formalin fixation
Staining mechanism for Perl’s Prussian blue
True HISTOCHEMICAL
What type of fixatives should be avoided when fat needs to be demonstrated ?
Anything with alcohol will extract lipids
What type of mounting media must be used for slides stained with Oil Red O ?
Aqueous mounting media; solvents in resinous media will dissolve lipids and Oil Red O
Which fixation artifact can cause a false positive result in FM ?
Formalin pigment; argentaffin
2 issues unbuffered formalin can cause when attempting to demonstrate calcium with Von Kossa’s Silver stain method
- Unbuffered formalin will have a lower pH = formalin pigment forms = false pos
- Low pH can dissolve calcium = false negative
Which endogenous pigment can interfere with Von Kossa’s Silver stain
Melanin
Which step is most crucial in Modified Gram staining ?
Differentiation/ decolorizing
What type of tissue is used as a control in Gram staining ?
Tissue with both gram pos and neg bacteria
Ie. placenta seeded with organisms, infected appendix..
If Gram control tissue is acceptable, but organisms in the patient sample are too pale, what might this indicate ?
- Patient may be on antibiotics
- Organism may be acid-fast
Why is it important not to miss acid-fast bacilli in a tissue section ?
- treatment differs from other infections (resistant to routine antibiotics)
- Mycobacterium are level 3 organisms = severe consequences on patient
How to fix a slide over stained with methylene blue in the Ziehl-Neelsten stain
- remove methylene blue completely with acid alcohol and re-counterstain
- organism should remain stained
How does the Giemsa stain differ from the Wright stain ?
- Romanowsky stains are very similar
Wright’s: polychromed Methylene blue and Eosin Y
Giemsa: Azure B and Eosin Y
- uses a more controlled oxidation method
- more reddish
- stain of choice for malaria
Besides Grocott’s methenamine silver, what other stains can be used to demonstrate fungi ?
- PAS
- Gram stain
- H&E
- Giemsa
- other metachromatic stains
Why is it important to know whether P. jirovecii or another fungal species is suspected ?
P. jirovecii takes longer to stain than other fungal organisms
What other organisms are silver methods used to demonstrate ?
Actinomyces, encapsulated bacteria, spirochetes
What staining mechanism is involved in Alcian Blue staining ?
A. Selective solubility
B. Ionic bonding
C. Covalent bonding
D. Porosity
B. Ionic bonding
When reviewing the control slide, some of the nuclei are staining with Alcian Blue. What is the most likely cause ?
Staining was greatly prolonged; only acid mucins should be stained
Why may apple-green birefringence in Amyloid-positive tissue not be seen ?
- sections cut too early (amyloid staining weakens with time after sections are cut)
- extended formalin fixation
- section too thick/ thin
Why are saturated salt solutions used in Congo red staining ?
To reduce non-specific staining
What is the recommended fixative for Congo red slides ? Why ?
Alcohol; formalin fixation decreases staining intensity of Amyloid
Which staining mechanism is involved in Congo red staining ?
Hydrogen bonding
What characteristic of amyloid explains its affinity for Congo red ?
Linear structure of Congo red fit within the secondary protein structure of Amlyloid (β-pleated sheets)
You notice that the melanin on your Fontana Masson control is stained weak. What is the most likely cause ?
Excess ammonia in your silver solution
Mechanism for Gordon and Sweet’s metallic impregnation method ?
Induced argyrophilia
Your control for Gordon&Sweet’s Reticulin stain is very weak and non-continuous. What is the most likely cause ?
- excess ammonia in silver solution
- neutral buffered formalin was used as the reducing agent (NON-BUFFERED FORMALIN MUST BE USED)
Which organism requires a longer impregnation step using Grocott’s Methenamine Silver ?
Pneumocystis jirovecii
The control slide for Grocott’s Methenamine Silver have fungi stained completely black, without internal structure. What is the most likely cause of this ?
Overstained in silver solution
Which characteristic of fungi is used to selectively “stain” with Grocott’s Methenamine Silver ?
Polysaccharide content
Why is chromic acid the oxidizer in Grocott’s Methenamine Silver ?
It over-oxidizes other tissue elements beyond aldehydes, excluding fungi cell wall
Which of the following statements apply to Oil Red O staining ? Select all that apply:
A. Demonstrates hydrophobic lipids
B. Demonstrates hydrophilic lipids
C. Avoid alcohol-containing fixatives
D. Must use aqueous mounting media
E. Heat staining solution to 65 degrees C
A. Demonstrates hydrophobic lipids
C. Avoid alcohol-containing fixatives
D. Must use aqueous mounting media
