Histotechnology HTL Exam - Comprehensive Review
Fuelgon Reaction
- Demo DNA, based on the hydrolysis of DNA - Fix in anything but Bouin's - Paraffin, 5um, no control all nuclei will give +rxn - Reagents: 1N HCl, Schiff's Reagent (DI water, basic Fuchsin, sodium metabisulfite, 1N HCl), Sulfurous Acid - HCl-hydrolysis, Schiff's-stain, sulfurous acid-diff, light green-counter Results: DNA-reddish purple
PAS Reaction
- Demo polysaccharides, neutral mucosubstances, and basement membranes -Oxidize certain tissue elements to aldehydes by periodic acid -Fix: 10% NBF or Bouins, blood smears fix in methyl alcohol -Paraffin 4-5um, kidney 1-2um, QC-kidney or liver if demo glycogen -Reagents- periodic acid, 1N HCl, Schiff's Reagent (DI water, basic Fuchsin, sodium metabisulfite, 1N HCl), potassium metabisulfite (removes excess stain) - can counter in Harris Hematoxylin -Results: Glycogen, neutral mucosubstances, sulfomucin, sialomucins, colloid material (thyroid, pars), basement membranes, fungal walls-PAS/Bright Rose reaction
Methyl Green-Pyronin Y
- Differentiate between DNA and RNA, ID plasma cells or immunoblasts in tissue - DNA stains with methyl green while RNA is stained red with pyronin - Fix 10% NBF, can also use B-5, Zenker's, and Helly's - paraffin 4um, QC-formalin-fixed section with plasma cells - Reagents: Methyl green-Pyronin Y (Acetate Buffer soln (acetic acid, sodium acetate), methyl green, pyronin Y) Results: DNA-green to blue/green, RNA-red, Goblet Cells-mint green, Background-pale pink to colorless, Immunoblast and plasma cell cytoplasm-intense red, Nuclei-green to blue/green
Brown-Hopps Modification of the Gram Stain
- demo gram + and gram - bacteria in tissues -Gram + bacteria wall is thicker, hold stain easier -Fix 10%NBF, paraffin 4-5um, QC-section with gram + & - -Reagents: Crystal Violet, Gram's Iodine, Basic Fuchsin Soln, Gallego's soln (diff), Picric acid-acetone -Results: Gram pos-blue, gram neg-red, background-yellow, nuclei- ligh red
May-Grunwald Giemsa Stain
- differentiate cells in hematopoietic tissue and demo microorganisms - Fix: Zenker's or B-5 preferred, but 10% NBF can be used -Paraffin 4-5um, QC-spleen - Reagents: Jenner Solution (Jenner dye, methyl alcohol, DI), Geimsa Solution (Giemsa, glycerin, absolute methyl alcohol, DI), Acetic water -Remove mercury pigments if necessary, methyl alcohol (prepare for stain), acetic water (diff) - Results: Nuclei-blue, cytoplasm of leukocytes- shades of pink, gray, or blue, Bacteria-blue
A cryostat is maintained at what temp
-20 C
What is the temp of: a cryostat
-20 C
What is the temp of: a freezer
-20 or -70 C
Van Geison's Picric Acid-Acid Fuchsin Stain
-Considered a primary connective tissue stain, used most as a counterstain (Verhoeff elastic technique) -Fix: any well fixed tissue, Paraffin 4-5um, QC-practically every tissue has internal control - Reagents: Wiegert's Iron Hematoxylin, Van Geison's Solution (Acid Fuchsin, Picric Acid) Results: Nuclei-black, Collagen-brilliant red, Muscle and cytoplasm-yellow
PAS Reaction with Diastase Digestion
-Demo Glycogen - Fix in 10%NBF, Formalin Alcohol, or absolute Alcohol -2 paraffin sections, one with and one without, 2 controls of liver containing glycogen -Reagents: periodic acid, Schiff Reagent, 1N HCl, diastase v. distilled water -Results: Glycogen-bright rose red on section "without" and absent on "with"
Von Kossa's Calcium Stain
-ID calcium in tissue -Fix: alcohols (preferred) or 10%NBF, Paraffin 4-5um, QC-section containing calcium -Reagents: Silver Nitrate, Sodium Thio, NFR, bright light (sunlight) is the reducer -Results: calcium salts-black, background-red
Alizarin Red S Calcium Stain
-ID calcium in tissue -calcium forms an Alizarin Red S complex in a chelation process -Fix in alcoholic formalin or 10%NBF, paraffin 4-5um, QC-a section containing calcium -Reagents: Alizarin red S Staining soln (alizarin red S, DI, adjust pH 4.1-4.3, with ammonium hydroxide) -Results: calcium deposit-orange red
Gomori's One-Step Trichrome Stain
-ID increase in collagenous connective tissue fibers, or diff between collagen and smooth muscle fibers -Plasma stain-chromatotrope 2R, Connective tissue fiber stain-Fast green, light green, or analine blue, phosphotungstic acid-stains muscle and cytoplasm red -Fix: any well fixed tissue, bouin's is used as a mordant to intensify color reactions, paraffin 4-5um, QC-every tissue has an internal control (can use uterus, small intestine, appendix or fallopian tube) -Reagents: Bouin's, Weigert's Iron Hematoxylin, Gomori's Trichrome stain ( Chromatotrope 2R; Fast Green FCF, Light Green, or Analine blue; Phosphotungstic Acid, GAA, DI), Acetic Acid Soln - Results: Nuclei-black, Cytoplasm, Keratin, and Muscle fibers-red, Collagen and mucus-green or blue
Potassium dichromate increases availability of which group for binding dyes?
-NH2
Formaldehyde crosslinks proteins by reacting with the
-NH2 group
If the pH of the staining solution is between 4.6 and 5.0, eosin combines with which tissue chemical group
-NH3+
Periodic Acid-Methanamine for Basement Membranes
-delineates the glomerular basement mem -Fix: 10%NBF (not mercury fix), paraffin 2um, QC-kidney has an internal control -Reagents: Methanamine Silver, periodic acid soln, Gold chloride, Light green -Results: basement mem-black, background-green
Alcian Blue, pH 2.5
-demo acid mucopolysaccharides -Fix in 10% NBF or Bouin's, paraffin 4-5um, QC-section of small intestine, appendix, or colon -Reagents: Acetic Acid, Alcian Blue, Nuclear Fast Red -Results: weakly acidic sulfated mucosubstances, hyaluronic acid, and sialomucins-dark blue, background-pink to red
Alkaline Congo Red Method
-demo amyloid in tissues (green bifringence after congo red staining) -Fix: alcohol or carnoy's preferred, 10% NBF, bouin's or zenker's can also be used, paraffin sections at 6-10um, QC-sections containing amyloid -Reagents: Stock 80% alcohol saturated with sodium chloride (NaCl, DI water, ethyl alcohol), alkaline salt solution (Stock 80%..., NaOH), Congo Red (Stock 80%..., congo red, NaOH) -Results: amyloid-deep pink to red, Elastic tissue- pale pink, nuclei- blue
Churkurian-Schenk Method for Argyrophil Granules
-demo argyrophil granules in neurosecretory tumors -Fix 10%NBF, paraffin 4-5um, QC-argyrophil positive carcinoid tumor is preferred, but a section of small intestine can bu used -Reagents: citric acid, acidified water, silver nitrate soln, reducing soln (sodium sulfite, hydroquinone, di), NFR -Results: argyrophil granules-black, argentaffin substances-black, nuclei-red, background- yellow brown
Grimelius' Argyrophil Stain
-demo argyrophil granules in neurosecretory tumors -Fix in 10% NBF, paraffin 4-5um, QC-argyrophil positive carcinoid tumor or small intestine -Reagents: silver soln (silver nitrate soln, acetic acid-sodium acetate buffer), Reducing soln (Hydroquinone, sodium sulfite, di water), NFR -Results: Argentaffin granules-dark brown to black, argyrophil granules-dark brown to black, nuclei-red, background-pale yellow
Cajal's Stain for Astrocytes
-demo astrocytes -Fix: formalin ammoniom bromide, frozen sections 20-30um, QC-cerebral cortex -Reagents: Formalin ammonium bromide, gold sublimate, sodium thio -Results: astrocytes with perivascular feet-black
Hall's Bile Stain
-demo bilirubin in tissue -Fix 10%NBF, paraffin at 4-5um or frozen sxn, QC-tissue containing bile -Reagents: Fouchet's Reagent (Trichloracetic acid, DI, Ferric Chloride), Van Geison's Soln-Acid Fuchsin, Picric Acid) -Results: bile or bilirubin-emerald green to olive drab, background-yellow
Luxol Fast Blue-Cresyl Echt Violet
-demo both myelin and Nissl substance -Fix 10%NBF, paraffin 10-15um, QC-section of spinal cord or medulla -Reagents: Luxol Fast Blue soln ( LFB MBS, alcohol, acetic acid), Cresyl Echt Violet (counter), Lithium Carbonate Solution (diff), 70% alcohol (diff 2-gray and white matter) -Results: Myelin-blue, nissl substance-violet, nuclei-violet
Luxol Fast Blue-Holmes' Silver Nitrate Method
-demo both myelin and nerve fibers -Fix 10%NBF, paraffin 10-15um, QC-section of cerebral cortex - Reagents: Aqueous silver nitrate, Impregnating soln (boric acid, borax, DI, silver nitrate, pyridine), Reducing soln (hydroquinone, sodium sulfite, DI), Gold Chloride (toner), Oxalic Acid (diff), Sodium Thio (remove xs silver), LFB, Lithium carbonate (diff) -Results: myelin sheaths-blue to green, axon and nerve fibers-black
Osmium Tetroxide Paraffin Procedure for fat
-demo fat, When osmium tet. chemically combines with fat it blackens it in the process -Fix in 10%NBF, paraffin 2um (stain the wet tissue block and then process starting in 70%) -Reagents: Osumium Tetroxide Solution (stain), Periodic Acid Solution (diff)-may be stained with H&E or Masson's Trichrome -Results: fat-black, other tissue elements: according to method used
Muller-Mowry Colloidal Iron
-demo carboxylated and sulfated mucopolysaccharides and glycoproteins -Fix: 10%NBF, Carnoy's, or alcoholic formalin preferred, avoid chromate fixatives; paraffin 4-5um, QC-small bowel, appendix or colon -Reagents: Working Colloidal Iron Solution (DI, Ferric Chloride, GAA), Ferrocyanide-hydrochloric acid (potassium ferrocyanide, HCl), acetic acid solution, nuclear fast red solution -acetic acid soln-prevents dilution of colloidal iron -Results: Acid mucopolysaccharides and sialomucins-deep blue, Nuclei-pink/red, Cytoplasm-pink
Grocott's Methenamine-Silver Nitrate Fungus Stain
-demo fungal orgs -Fix:10%NBF, paraffin 4-5um or 6um frozen, QC- section containing fungi -Reagents: chromic acid(ox), Methanamin-silver nitrate soln (silver nitrate, methenamine soln, borax soln, DI), Sodium bisulfite (remove chromic acid), gold chloride (toner), Sodium thio (remove unreduced silver), Light green (counter) -Results: fungi-sharply delineated in black, mucin-taupe to dark gray, background-green (can be done rapidly using frozen sections fixed in 40% formaldehyde-useful in the diagnosis of P. Carinii-which stains black)
Hotchkiss-McManus PAS Reaction
-demo fungi -Fix 10%NBF or Bouin's or Zenker's, paraffin 4-5um, QC-sxn with Fungi -Reagents: Periodic acid, Schiff reagent, Sulfurous rinse soln (DI, 1N HCl, sodium metabisulfite), 1:5,000Fast Green Soln (FG, DI, GAA) -Results: fungi-rose, background-green
Holzer's Method
-demo glial fibers -fix 10%NBF, paraffni 6-8um, QC-section of cerebral cort -Reagents: 0.5% phosphomolybdic acid-alcohol soln, Crystal violet stain (abs alcohol, chloroform, stain), Potassium bromide soln (stain), differentiating soln (aniline oil, chloroform, ammonium hydroxide) -Results: glial fibers-blue -crystal violet may be removed with aniline oil
Toluidine Blue for Mast Cells
-demo mast cells -metachromatic stain-diff color than dye -fix 10%NBF, paraffin 4-5um, QC-sxn containing mast cell -Reagents: Toluidine Blue -Results: mast cells-deep violet, background-blue
Mallory's Phosphotungstic Acid-Hematoxylin
-demo muscle cross-striations, fibrin, and may demo Nemaline Rods (cross-striations diagnose rhabdomyosarcomas-tumors from striated muscle), also demo glial fibers -Polychrome stain -Fix: Zenker's preferred but 10%NBF may be used, paraffin 4-6um, QC-skeletal or cardiac muscle to demo cross-striations, sxn with fibrin, or section of cerebral cortex for glial fibers (paraffin 6-8um) -Reagents: Phosphotungstic Acid- Hematoxylin (PTAH), Gram's Iodine (lugol's for glial fibers), Sodium Thiosulfate, Potassium Permanganate, Oxalic Acid Results: Cross striations, fibrin-blue, nuclei-blue, collagen-red/brown, Neurons-salmon, Glial fibers and Myelin-blue
Weil's Method
-demo myelin -regressive stain -Fix 10%NBF, paraffin 10-15um, QC-spinal cord or medulla -Reagents: Ferric Ammonium sulfate (diff), Alcoholic hematoxylin, differentiating soln (sodium borate, potassium ferricyanide) -Results: Myelin Sheath-blue to blue/black
Luxol Fast Blue
-demo myelin in tissue -Fix 10%NBF, paraffin 10-15um, QC-spinal cord or medulla -Reagents: Luxol Fast Blue, lithium carbonate soln (diff), 70% alcohol(diff step 2-grey v white matter) -results: myelin-blue
Holmes' Silver Nitrate Method
-demo nerve fibers and neurofibrils -argyrophil rxn-requires that a chemical reducer be used -Fix: 10%NBF, Paraffin 10-15um, QC-sxn of cerebral cort -Reagents:Aqueous silver nitrate, Impregnating soln (boric acid, borax, DI water, silver nitrate, pyridine), Reducing soln (hydroquinone, sodium sulfite, DI), Gold Chloride (toner), Oxalic Acid (reduce gold), Sodium Thio (remove XS silver) -Results: axons,nerve fibers, and neurofibrils-black
Bodian's Method
-demo nerve fibers in tissue sections -fix 10% NBF, paraffin 6-8um, QC- sxn of peripheral nerve or cerebral cortex -Reagents: Protargol Soln (Silver impregnator), Reducing soln (hydroquinone, formaldehyde), Gold chloride (toner), Oxalic Acid (reduce gold), Sodium Thio (Remove unreduced silver), Aqua Regia (HCl, Nitric Acid)-cleans copper, Aniline blue (counter), copper is added to silver soln to destain connective tissue -Results: Nerve fibers-black, background-blue, nuclei-black
Nissl Substance: Cresyl Echt Violet Method
-demo neurons and loss of Nissl substance -Nissl substance-in neurons, composed of RER -Fix 10% NBF, paraffin 6-8um, QC-section of spinal cord -Reagents: Cresyl Echt Violet soln, Balsam-Xylene Mix, Absolute Alcohol (diff) -Results: Nissle Substance-blue to purple, background-colorless -this stain can also be done at an acid pH, restricting staining to DNA and RNA containing strx (no Balsam-Xylene mix)-appears unstained macroscopically
Sudan Black B
-demo neutral lipids -more sensitive lipid dye, soluble in phospholipids, also used in hematopathology to diff granulocyte precursors from leukocytes -Fix: 10% NBF or sections post-fixed in calcium formol (no alcohol), frozen at 10um, QC-most tissue contains fat (none) -Reagents: Calcium Formol, Sudan Black B (stain, propylene glycol), Propylene glycol (sensitize, differentiate), NFR (counter) -Results: Fat-blue/black, Nuclei-red
Oil Red O Method (neutral fats)
-demo neutral lipids in tissue -Fix: 10%NBF or calcium formal (no alcohol), frozen sections at 10um, QC-most tissue contains some fat so normally a control is not used -Reagents: Oil red O (stain, isopropanol, DI), Harris Hematox w/ acetic acid (counter), Ammonia water (blue), mount with aqueous media Results: fat-intense red, nuclei-blue
Gridley's Fungus Stain
-demo of fungi in tissue -Fix 10%NBF, paraffin 4-5um, QC-sxn with fungi -Reagents: Chromic acid (oxidizer), Schiff reagent, Aldehyde Fuchsin stain (basic fuchsin, 70% alcohol, HCl, paraldehyde), Metanil Yellow (counter) -Results: mycelia-deep purple, conidia-deep rose to purple, background-yellow, elastic fibers and mucin-deep purple
Aldehyde Fuchsin Stain
-demo pathologic changes in elastic fibers -Fix-any well-fixed tissue, paraffin 4-5um, QC-a section of aorta embedded on edge, cross section of a large artery, or skin -Reagents: Aldehyde Fuchsin Stain(Pararosaniline, alcohol, HCl, paraldehyde), Light Green (Light Green SF yellowish, GAA, DI) -Results: Elastic Fibers-deep blue to purple, other tissue elements-green
Fontana-Masson Stain for Melanin and Argentaffin Granules
-to demo argentaffin substances (ie melanin, argentaffin granules of carcinoid tumors, and some neurosecretory granules) -Fix in 10% NBF, alcohol should be avoided because it dissolves argentaffin granules,paraffin 4-5um, QC-section of skin of melanin and a section of small intestine or appendix as a control for argentaffin granules -Reagents: Fontana's Silver solution (silver nitrate ammonium hydroxide), Gold Chloride solution, Sodium Thio, NFR -Results: melanin-black, argentaffin granules-black, nuclei-pink
The preferred fixative for the warthin-starry stain is
10 % NBF
Which fixative is considered the "routine fixative"?
10% NB Formalin
Fixative for PPB is
10% NBF
Fixative used for Alcian blue with hyaluronidase
10% NBF
Fixative used for Mayer mucicarmine stain
10% NBF
What fixative should be buffered before use
10% NBF
What is the fixative for: Aldehyde fuchsin
10% NBF
What is the fixative for: Gomiri Trichrome
10% NBF
What is the fixative for: Gomori reticulin
10% NBF
What is the fixative for: Oil Red O
10% NBF
Fixative to use with Alcian blue 1.0 is
10% NBF or Bouins
Fixative for PAS is
10% NBF or Bouins solution
Fixative for Alcian blue PAS hematoxylin
10% NBF or zenker solution
Alcian blue, pH 2.5 fixative
10% NBF, Bouin
What is the fixative for: Verhoeff-van Gieson
10% NBF, Zenkers
PAS fixative for glycogen
10% NBF, formalin alcohol or absolute alcohol
What is the fixative for: Phosphotungstic acid-hematoxylin
10%NBF, Zenkers
The preferred micrometer thickness for sections to be stained by the technique shown in the image is
10-15
Sections to be stained for myelin should be cut at
10-15 nm
The sections for the technique shown in the image should be cut at
10-15nm
A technologist completed an H +E stain on breast tissue and decided to check the slide under the microscope to verify if the stain worked correctly. The technologist noticed that all the lipids were destroyed in the tissue. What fixative was used in the grossing area that could of destroyed all the lipids on the slide?
100% Ethanol
How long does it take for processor to complete the cycle
13 hrs
The volume of fixative should exceed the volume of the tissue by
15-20 times
The volume of fixing fluid should be
15-20 times th amount of specimen
Quality control for PAS glycogen stains require
2 control sections of liver containing glycogen (1with diastase 1 without)
Sections for the demo if basement membranes should be cut at
2 nm
Control tissue for Alcian blue with hyaluronidase
2 sections of umbilical cord (with and without) Section of small bowel, appendix or color may be used as a second control to demonstrate epithelial mucins
Evaluation of the stain shown in the image reveals
2 shades of eosin staining
Sections for the technique in the image should be cut at
2-3 nm
When used in a (___) Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins)
3% acetic acid solution (pH 2.5)
This is highly sensitive for lipid but it is not permanent in tissue sections
3,4-Benzpyrene
The clearance angel of the mictotome blade is routinely
3-8
The clearince angle on a rotary microtome is
3-8*
The regular laboratory incubator maintains a temperature of about
37 C
What is the temp of: a lab incubator
37 C
Commercial stock formaldehyde solutions contain
37-40% formaldehyde
The ordinary refrigerator, when opening normally, has a temperature that approaches
4 C
What is the temp of: a refrigerator
4 C
10% formalin is the same as
4% formaldehyde
10% formalin is equal to
4% paraformaldehyde is equal to
For the best cytoplasmic staining, the pH of the eosen should be between
4.6 and 5.0
Commercial stock formaldehyde solution contains:
40% formaldehyde
When using a microscope with a X10 ocular and a X40 objective, the total magnification is approximately
400
Temperatures used for drying section
45 55 37
What is the temp of: a flotation bath
45 C
When using paraffin with a melting point of 55-57 C, the most common temp for floating sections on a water bath is approx
45-50 C
Melting point of soft wax
45-50*C
Storage for Schiff reagent
4C, up to 3 months
Size of tissue cassets
4x3x0.5 cm
Formalin pigment would most certainly be formed when the pH of the formalin is
5.0
Melting point of Hard wax
50-60-*C
After fixing in Bouins, the excess picric acid is frequently removed by washing in
50-70% alcohol
H&E stained sections of liver fixed in the microwave oven show marked pyknotic, overstained nuclei. This can probably be prevented in the future by ensuring that the temperature is kept below
55 C
A good paraffin got routine use is one with a melting point of
55-58 C
See Schedule: Formalin, 10% 2 hrs (no heat, vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) 95 % alcohol 1 hr (only vacuum) 95 % Alcohol 45 min (no heat, no vacuum) Absolute Alcohol 45 min (only vacuum) Absolute Alcohol 1 hr (no heat, no vacuum) Xylene 1 hr no heat, no vacuum) Xylene 1 hr (only vacuum) Paraffin 30 min (no vacuum) Paraffin 1 hr (no vacuum) Paraffin 1.5 hr (vacuum) If the above schedule began at 5 pm, the tissue would be ready for embedding at what time
5:30 am
The recommended minimum time for fixation in formalin is
6-8 hours
The Giemsa stain is most satisfactory if the pH os between
6.4 and 6.9
Most commonly, the paraffin used for embedding tissues is kept at approximately
60 C
The temperature of the oven used to maintain a supply of melted paraffin for embedding tissue is most commonly at
60 C
What is the temp of: an infiltrating paraffin container
60 C
thickness of speciments using cryostat
6um store at -70 *C
After fixing tissue in Bouin's solution the excess picric acid is removed by washing in:
70% Ethanol
Section thickness should be this for lipids
8-10
Biopsy processing example
80% alcohol 40min (2 changes total) 95% alcohol 40 min (2 changes total) Absolute alcohol 60 min (2 changes total) Xylene 40 min (2 changes total) Paraffin 40 min (2 changes total)
Other glycogen fixatives
80% alcohol, alcoholic formalin (37% formalin, 95% alcohol), acetic alcohol formalin, aqueous solution (formalin)
Epoxy resins are cut to what thickness on a microtome
80-90 nm
Alcian blue suffix
8GX
To prepare a 10% solution of formalin, how much water should be added to 100mL of stock formaldehyde?
900 mL
The ratio of stock solution to acid in Zenker fixative is
95 parts stock to 5 parts acetic acid
Which fixative is classified as a non-additive
95% Ethyl alcohol
What is in: Gendre
95% alcohol, acetic acid, formaldehyde, picric acid
What stain is a silver impregnation method
Holmes
What stain will demo nerve fibers
Holmes
What stain will demo astrocytic processes
Holzer
What stain will demo gliosis
Holzer
Many histologist prefer to use this, which contains only alpha amylase for digestion or glycogen
Human saliva
Staining will disappear or be dramatically reduced when tissue sections containing (Alcian blue 1.0)
Hyaluronic acid, chondroitin sulfate A or C (connective tissue mucin) are digested with testicular hyaluronidase
Connective tissue mucins include
Hyaluronic acid, chondroitin sulfate A&C
Name a substance that is often combined with the Alcian blue technique
Hyaluronidase
Best carmine methods mechanism of action is
Hydrogen bonding
Mechanism of action of best carmine
Hydrogen bonding( phenol groups ionize to the oxide and bind to the glycol groups of glycogen)
What are the reagents in: Steiner and Steiner
Hydroquinine
The problem shown in the image is most likely due to
Improper embedding
The nuclear problem seen in the image is most commonly due to
Incomplete fixation
The problem shown in the image is
Incorrect orientation of the tissue
Dehydrant, clearing, or infiltration/embedding: araldite
Infiltration/embedding
Dehydrant, clearing, or infiltration/embedding: carbowax
Infiltration/embedding
Dehydrant, clearing, or infiltration/embedding: cellodin
Infiltration/embedding
The small square to the left of the surgical number in the cassette ID area in the image is most helpful for
Interfacing with computer and instrument info systems
Alcian blue PAS hematoxylin is used for the detection of
Intestinal metaplasia
Precipitate left in tissues that have been fixed in solutions containing mercuric chloride may be removed by immersion in
Iodine
What is the oldest method for carbohydrate staining?
Iodine method
Which type of metal salt serves as the mordant in Weigerts hematoxylin
Iron
Ammonium hydroxide is both a
Irritant and corrosive used in the hood
What solvents do not dissolve out lipids
Isopropanol glycol
Lipids in crystalline form are
Isotropic and birefringent
The pigment in the image could have been prevented by
Keeping the pH of the fixative >6.0
The tissue demoed in the image is
Kidney
The tissue shown in the image is
Kidney
For neutral mucins stain use these for controls (PAS)
Kidney , stomach
Order of gross the specimen
Kidney - Liver- Kidney - liver
The nuclear problem seen in the image is
Lack of chromatin definition
A dye appears colorless because its chromophore was reduced. The reaction is reversible.
Leuco compound
A colorless compound also known as
Leucofuchsin
Schiff reagent causes a reduction of quinoid structure resulting in
Leucofuchsin
If fungi are to be demoed, a good counterstain for the PAS technique is
Light green
List three type son microscopy most associated with histology
Light, electron, and polarized
The chief objection to the Usenet xylene as a clearing agent for processing g tissues is that xylene is
Likely to harden tissue
The component stained red in the image is
Lipid
Differentiating agents in the Luxol Fast Blue procedure for myelin are:
Lithium carbonate and 70% alcohol
A good control for glycogen is
Liver
Best carmine quality control tissue
Liver
The control used for the method in the image is
Liver
The tissue shown in the image is
Liver
Glycogen is most abundant in
Liver, heart (cardiac muscle cells), womb
The blue staining in the image is due to
Luxol Fast Blue
What technique uses copper phthalocyanine chromagen to stain tissue?
Luxol Fast Blue
What is the procedure for this reagent: Pyronin
MGP
This is commonly used for digestion but tends to loosen the sections and does not always completely digest the glycogen
Malt diastase
With digestion mucins containing hyaluronic acid and chondroitin sulfate A&C will show
Marked loss of staining
The tissue shown in the image was most likely stained by what method
Masson
What stains muscle red
Masson trichrome
What is the procedure for this reagent: Acid fuchsin
Masson trichrome, van Gieson
What technique uses light green to stain collagen?
Masson's Trichrome
Which staining technique uses Bouin's to increase acidophilia?
Masson's Trichrome trchnique
The cells with the rose-violet cytoplasm shown in the image are most likely
Mast cells
What tissue component has granules that stain metachromatically
Mast cells
The problem in the image probably would not occur if the aluminum hematoxylin used had been that of
Mayer
Which hematoxylin is most frequently used as a progressive stain
Mayer Hematoxylin
Which hematoxylin is most preferred for IHC stains
Mayer Hematoxylin
Name two hematoxylins that contain ammonium or potassium aluminum sulfate
Mayer and Harris
This stain is used to demonstrate epithelial mucin in tissue sections
Mayer mucicarmine
A Hematoxylin solution that is commonly used progressively and very rarely used regressively is:
Mayers
B-5 Fixative Ingredients
Mercuric Chloride, Sodium Acetate, DI water, Formaldehyde (beautiful nuclear detail, must be treated to remove mercury pigment, preferred fixative for many Ab used in immunoperoxidase staining)
Zenker solution
Mercuric chloride potassium dichromate sodium sulfate (optional) distilled water acetic acid, glacial - lysis RBC recommended for subsequent AFB staining can decalcify needle biopsy of bone marrow
Helly solution
Mercuric chloride potassium dichromate sodium sulfate (optional) distilled water formaldehyde (Add just before use because it is a reducing agent and will cause the solution to become dark and turbid)
This type of fixative should be avoided for glycogen fixative due to?
Mercuric chloride fixatives (zenkers and helly)
The 3 ingredients in B5 fixative are:
Mercuric chloride, Sodium acetate, Formaldehyde
___________Increses Acidophilia and basophilia of the tissue
Mercury fixative
This removes excess Schiff reagent
Metabisulfite
Another technique that could be used to demo the same tissue component that is stained rose in the image is
Methenamine silver
Fixative for PAS for blood smears should be
Methyl alcohol
To prevent polymerization of formaldehyde, what is added to commercial stock solutions
Methyl alcohol
What stain demos DNA
Methyl green-pyronin
What occures when additive fixative is used
Methylene bridges
The phenomenon that occurs when a single pure dye stains chromotropic tissue in a color that differs from that of the dye solution (due to polymerization).
Metochromasia
Satisfactory electron microscopy can be obtained on a specimen from the wet tissue file, if it has been fixed and stored in
Millonig formalin or formaldehyde-glutaraldehyde (4CF-1G)
What is the function of: iodine
Mordant
What is the function of ferric chloride in the Verhoeff's Van Gieson technique?
Mordant Oxidizer Differentiator
The technique shown in the image is most likely that of
Movat
The problem seen in the image could most likely be corrected by
Moving to an unused part of the blade
The issue in the image can be corrected by
Moving to an unused section of the blade
Build up of this in certain inflammations, increase in certain intestinal carcinomas
Mucin
Function of these...; lubrication, coat cell surface to provide favorable environment for ionic and molecular diffusion, provide greater adhesion between adjacent cells (glue)
Mucin
The results of Mayer's mucicarmine stain
Mucin, capsule of cryptococcus (neoformans) = deep rose to Red, nuclei = Black, background blue or yellow
For Skeletal muscle list the # and location of nuclei, is it striated, is it voluntary
Multiple, peripherally located nuclei, striated, voluntary
Which layer of tissue is missing from the image shown
Muscularis externa
What is stained by: Auramine - rhodamine
Mycobacterium tuberculosis
What is stained by: Ziegler's-Neelsen
Mycobacterium tuberculosis
What is found on the mycobacterium cell wall that allows staining with carbol fuchsin to occur?
Mycolic acid
What is the tissue component is demoed by: Borax-ferricyanide
Myelin
What is the tissue component is demoed by: Iron hematoxylin
Myelin
What is the tissue component is demoed by: phosphotungstic acid hematoxylin
Myelin and glial fibers
The preferred fixative for the Bodian technique is
NBF
The preferred fixative for the technique in the image is
NBF
What type of resin causes fading of stains with prolonged storage
Natural
name a resin that yellows as the preparation ages
Natural resin
Basophilic tissue has a ___________ charge.
Negative
For best results when using formalin as a routine fixative, it must be made
Neutral
Group 1 carbohydrates are
Neutral polysaccharides (nonionic homoglycans)
Before processing decalcified tissue, what is the next step?
Neutralize acid
This staining method is useful to demonstrate both neutral and acidic lipids
Nile Blue Sulfate
Acidic and neutral lipids can be distinguished by
Nile blue sulfate
Cresyl Echt Violet demonstrates
Nissl substance
What is the tissue component is demoed by: Cresyl echt violet
Nissl substance
Acetic Acid
Non-coagulant (only nucleoprotein), does not fix carbs or lipids, dissolves out certain cell organelles (mito and golgi), precipitates DNA, lyses RBCs
Crooked paraffin ribbons may be caused by
Nonparallel block horizontal edges
The nuclear problem seen in the gland in the image is
Nuclear bubbling
The counterstain used in the technique shown in the image is most likely
Nuclear fast red
When staining tissues with Verhoeff's Van Gieson (VVG), what is the appropiate colors for the tissue components as an end result when stain is complete.
Nuclei = black Collagen = red Reticulin = not demonstrated Elastin = black Cytoplasm = yellow Muscle = yellow RBC's = bright yellow Lipids - no
When staining tissues with Hematoxylin and Eosin (H+E), what is the appropiate colors for the tissue components as an end result when stain is complete.
Nuclei = blue Collagen = pink Reticulin = not demonstrated Cytoplasm = pink Muscle = pink RBC's = dark pink Lipid = not demonstrated
When staining tissues with Masson's Trichrome (MT), what is the appropiate colors for the tissue components as an end result when stain is complete.
Nuclei= black Collagen= blue/green Reticulin= not demonstarted Elastin= red Cytoplasm= red Muscle= red RBC's= red Lipids= not demonstrated
When staining tissues with Gordon and Sweets (G&S), what is the appropiate colors for the tissue components as an end result when stain is complete.
Nuclei= red Collagen= pink/grey Reticulin= black/grey Elastin= pink Cytoplasm= pink Muscle= pink RBC's= pink Lipids= not demonstrated
When staining tissues with Gomori Aldehyde Fuchsin (GAF), what is the appropiate colors for the tissue components as an end result when stain is complete.
Nuclei= yellow Collagen= red Reticulin= not demonstrated Elastin= purple Cytoplasm= yellow Muscle= yellow RBC's= bright yellow Lipids= not demonstrated
This is useful for demonstrating both hydrophobic and hydrophilic lipids
OTAN
This technique distinguishes from Hydrophobic and hydrophilic lipids
OTAN
The pigmented the image most likely
Occurred during fixation with acidic formalin
The issue in the image is most likely the result
Of cross contamination during deparaffinization
A dye that is not water soluble
Oil Red O
The technique in the image is
Oil Red O
List a technique that is an example of dye absorption
Oil red O
What tissue component is used to demo adipose cells
Oil red O
Which of the following methods is an example of physical staining: toluidine blue verhoeff-van gieson methenamine silver oil red o
Oil red O
name a stain that requires mounting with an aqueous mounting medium
Oil red O
ATPase Enyme Histochemistry demonstrates
ATPase in muscles
For Smooth muscle list the # and location of nuclei, is it striated, is it voluntary
One centrally located nuclei, not striated, not voluntary
For Cardiac muscle list the # and location of nuclei, is it striated, is it voluntary
One centrally located nuclei, striated, not voluntary
The problem shown in the image could have been prevented by
Opening specimen, pinning it out, and adding fixative upon receipt
Lipids are soluble in
Organic solvents
What's the fixative for: Pheochromocytomas
Orth solution
The staining of tissue, with a dye, in a predictable way. A green dye stains the tissue green. ____________
Orthochromasia
The grossing area receives a liver biopsy that appears to be fatty. The pathologist wants to preserve the lipids in the tissue. What is the best fixative to use for lipid studies?
Osmium Tetroxide
Fats are usually preserved best if the tissue is fixed in
Osmium tetroxide
Name a fixative that will leave the tissue protein uncoagulated
Osmium tetroxide
Neutral lipids are chemically fixed and made insoluble in tissue by:
Osmium tetroxide
Which fixative makes lipids insoluble?
Osmium tetroxide
The problem shown in the image is most frequently the result of
Over dehydration
The problem in the image is most likely due to
Overdecalcification
Microscopic examination of an H&E stained section reveals marked chatter, especially at the edges of the tissue. This was most likely caused by
Overdehydration of the tissue
When cutting paraffin embedded tissue, of the tissue seems hard and brittle, one source of trouble is likely to be
Overheated paraffin
What is the name of the bleaching agent in Gordon and Sweets (G+S)?
Oxalic acid
What is the function of: Periodic acid
Oxidation
What is the function of: chronic acid
Oxidation
Another technique for demoing the tissue component stained black in the image is
PAS
Staining mechanism of this stain: positive reaction depends on presence of 1,2 glycol grouping. Oxidation of certain tissue elements to aldehydes by periodic acid.
PAS
The staining technique shown in the image is
PAS
Uses an amylase to remove glycogen from tissue
PAS
What is a method for demoing fungi
PAS
What stain demos neutral polysaccharides
PAS
What stain is used for: neutral polysaccharides
PAS
What stain may give false positives after gluteraldehyde fixation
PAS
See Schedule: Formalin, 10% 2 hrs (no heat, vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) 95 % alcohol 1 hr (only vacuum) 95 % Alcohol 45 min (no heat, no vacuum) Absolute Alcohol 45 min (only vacuum) Absolute Alcohol 1 hr (no heat, no vacuum) Xylene 1 hr no heat, no vacuum) Xylene 1 hr (only vacuum) Paraffin 30 min (no vacuum) Paraffin 1 hr (no vacuum) Paraffin 1.5 hr (vacuum) If the above schedule was started at 5 pm and the power went out at 11:45 pm, what step would the processor be on
Absolute Alcohol
Carnoy's Solution Ingredients
Absolute Ethyl Alcohol, Chloroform, GAA (Lyse RBCs, preserve glycogen, good nuclear preservation, excessive shrinkage and hardening, fix no longer than 4 hrs) Methacarn substitutes methyl for ethyl alcohol, and it hardens and shrinks less than Carnoy's
H&E stained sections fail to reveal acid crystals on a case with clinical diagnosis of gout. One possible explanation for the false negative result could be that the specimen was fixed in a solution other than
Absolute alcohol
The preferred fixative for best carmine
Absolute alcohol
What is the best fixative for glycogen
Absolute alcohol
What is in: Carnoy
Absolute alcohol, acetic acid, chloroform
Used in staining techniques to increase the intensity and selectivity or affinity of dyes for certain tissues or components of tissues (example: phenol in carbol fuchsin).
Accentuator
_______counteracts the shrinking of the cells that occurs from picric acid in Bouins fixative
Acetic acid
What is in: Hollande
Acetic acid, copper acetate, formaldehyde, picric acid
What is in: Bouin
Acetic acid, formaldehyde, picric acid
What is in: Zenker
Acetic acid, picric acid, potassium dichromate
What are the reagents in: Brown and Hopps
Acetone, crystal violet, and picric acid
Which statement is true for Masson's Trichrome technique?
Acid Fuchsin initially stains the collagen red
What are the reagents in: Kinyoun
Acid alcohol
Ziehl Neelsen shows
Acid fast bacilli (T.B.)
What is a component of the Masson trichrome stain
Acid fuchsin
What is a component of the van Gieson stain
Acid fuchsin
What pigment can be prevented by using a neutral pH
Acid hematin
Results for AB PAS
Acid mucin = blue, neutral mucin = bright pink
Alcian Blue demonstrates
Acid mucins
Colloidal ironies used for the demo of
Acid mucopolysaccarides
Due to demonstrating epithelial mucins, Mayer's mucicarmine is used in the identification of
Adenocarcinomas
List two types of connective tissue cells
Adipose and mast cells
What type of tissue component requires frozen sections
Adipose tissue
Control tissue for fat
Adrenal
The problem shown in the image is the result of
Air trapped under the section
What stain is used for: acid mucopolysaccarides
Alcian Blue 1.0 and 2.5, colloidal iron, mucicarmine
What stain is sometimes used after hyaluronidase
Alcian Blue pH 2.5
Acid mucopolysaccharides are demoed by
Alcian blue
This dye is a copper phthalocyanin basic dye that is water soluble and colored blue because of its copper content
Alcian blue
The demonstration of sulfated mucosubstances is possible with this stain
Alcian blue 1.0
What stain is used for: Sulfated (only) acid mucopolysaccarides
Alcian blue 1.0
What stain demos acid mucosubstances
Alcian blue 2.5
What stain is used for: carboxylated acid mucopolysaccarides
Alcian blue 2.5 and colloidal iron
Which dyes stain weakly sulfated goblet cells? (3)
Alcian blue 2.5, metachromatic pH 2.0, colloidal iron
This stain is used to differentiate between neutral and acidic mucins
Alcian blue PAS hematoxylin
What stains demo sulfated sialomucins
Alcian blue pH 1.0 and 2.5
What substances demonstrate sulfated acid mucopolysaccarides
Alcian blue pH 1.0 and 2.5
Another technique that would look almost identical to the technique shown in the image is
Alcian blue pH 2.5
Of the following the technique shown in the image is most likely Schmorl technique for reducing substances PAS Alcian blue pH 2.5 crystal violet
Alcian blue pH 2.5
This stain is used to identify acid mucins
Alcian blue pH 2.5
What substance demos carboxylated acid mucopolysaccarides
Alcian blue pH 2.5
What is a superior alternative to Alcian blue 8GX due to the decrease in dye content and poorer dye solubility than when originally tested
Alcian blue pyridine variant
When using this only sulfated acid mucins and sulfated sialomucins (glycoproteins) are stained
Alcian blue when used in 0.1N HCL solution (pH 1.0)
This stain is used to differentiate epithelial from connective tissue mucins
Alcian blue with hyaluronidase
Neutral mucins are negative with these stains (3)
Alcian blue, colloidal iron and mucicarmine
Name a common dehydration agent
Alcohol
Non-additive fixatives ("a" words)
Alcohol, acetic acid, Acetone
Formalin pigment (AFH), may be removed from tissue by: (acidic ph and blood cause formalin pigment)
Alcoholic picric acid
Formalin pigment may be removed from tissue by
Alcoholic picric acid
The pigment seen in the image could most likely be removed by treating with
Alcoholic picric acid
Non-additive Fixatives
Alcohols and Acetone
After completing Gomori Aldehyde Fuschsin technique for elastic, you notice no elastic fibers are stained on your control slide. What is the reason for this?
Aldehyde Fuchsin is too old Aldehyde Fuchsin used too early and didn't give enough time to ripen Timing in Aldehyde Fuchsin solution is too short
What is the procedure for this reagent: Basic fuchsin
Aldehyde fuchsin
What is the procedure for this reagent: Paraldehyde
Aldehyde fuchsin
What stain uses paraldehyde in the primary stain
Aldehyde fuchsin
The schiff reaction demos
Aldehydes
The problem in the image can mostly likely be prevented by
Allowing the flotation bath water to stand overnight
The problem shown in the image could have been prevented by
Allowing the slide to drain well before drying at 60C
When performing Ziehl Neelson technique, Carbol Fucshin is heated at 60C. What would the heat do to the stain?
Allows for better dye penetration
Malt diastase contains both
Alpha and beta amylase
This metal is believed to form a chelation complex with the carmine, the resulting compound has a net positive charge and attaches to the acid groups of mucins
Aluminum
Which of the following is the mordant salt used to make Hematoxylin solutions most commonly used in the routine H+E?
Aluminum
Counterstain used in PAS
Aluminum hematoxylin (common), working light green
The structure in the center of the image indicates
Alzheimers
Formaldehyde reacts with protein side chains by combining with the
Amino group
What chemical is used in the prep of Harris hematoxylin
Ammonium aluminum sulfate
hematoxylin solutions with this chemical are considered stable
Ammonium aluminum sulfate
what chemical acts as a mordant in hematoxylin solutions
Ammonium aluminum sulfate
what chemicals serve as mordants in hematoxylin solutions
Ammonium aluminum sulfate and ferric chloride
Delafield's Hematoxylin
Ammonium aluminum sulfate, DI Water, hematoxylin, 95% alcohol, glycerol...Age 3-6 months mordant=aluminum, oxidation is natural exposure to light and air, Alcohol=preservative, glycerol=protection from overoxidation (used regressivley)
List the two solutions for differentiation in the Weil procedure
Ammonium ferric sulfate, Borax ferricyanide
The substance demoed in the image is
Amyloid
The substance demoed in the image is most likely
Amyloid
The issue in the image would not result from
An extended block holder shaft
The issue in the image is most likely due to
An improperly clamped block
The problem in the image is most likely due to
An improperly dried slide
What stain reacts with aldehyde groupings
PAS
Staining reactions for glycogen:
PAS (+), diastase (sensitive), Bauer-feulgen (+), Best carmine (+)
Mucin is
PAS (+), metachromatic, basophilic
Glycogen, neutral mucins, certain epithelial sulfomucins and sialomucins, colloid material of thyroid and pars intermedia of the pituitary, basement membranes and fungal walls show positive with
PAS reaction
This stain is used for demonstration of glycogen in tissue sections
PAS reaction with diastase digestion
The best carmine technique is not as specific as this method, not as much glycogen demonstrated
PAS w/ or w/out diastase
Glycogen is best demonstrated by the use of
PAS with and without diastase
Polymerized formaldehyde is known as
Paraformaldehyde
Zamboni's Solution Ingredients
Paraformaldehyde, Picric Acid, DI Water, Phosphate buffer, NaOH to alkanilize the solution (pH 7.3) (Good general purpose, allows secondary fixation with osmium, good for EM)
What is in: Zamboni
Paraformaldehyde, Picric acid, sodium phosphate di and monobasic
Periodic acid is used in the PAS technique as
An oxidizer
After completion of a Masson's Trichrome technique, the cytoplasm appears "muddy". What is the cause of this result?
Aniline blue is causing a discoloration of the cytoplasm
Immunohistotochemistry
Anything an antibody can be made for
A good control for the Mayer mucicarmine stain is
Appendix
A substance that has the ability to bind to silver solution and reduce it to a metallic form is said to be:
Argentaffin
What does the ammonia responsible for in best carmine
As a solvent for carmine to help maintain the high pH
The black structures seen in the image are
Astrocytes
What is the tissue component is demoed by: Gold sublimate
Astrocytes
What is the tissue component is demoed by: Crystal violet
Astrocytes and glial fibers
The problem in the image most likely occurred
At embedding
Is the tissue in the image autolyzed or well preserved?
Autolyzed
It is important to not use this type of control material for Mayer mucicarmine
Autolyzed tissue
The ionizing radical of a dye which is responsible for its binding to oppositely charged tissue groups. (Examples: -OH, -SO3H, -COOH, -NH2).
Auxochrome
Sections in what fixative must be treated with iodine
B-5
Describe the choice of fixative to use and three reasons why you chose that fixative for immunological studies.
B5 (or Zinc Formalin) - Enhances immunoreactivity - Coagulant fixative - gives greater permeability - better antibody penetration - more intense staining - Enhances nuclear detail, and good morphology - Enhances staining of both nuclei & cytoplasm
Putrefaction of tissue is caused by
Bacterial action
Neutral mucins found in
Basement membrane, thyroid and stomach
Neutral mucin present in
Basement membrane, thyroid colloid, surface epithelium of stomach (goblet cells),
What is a component of aldehyde fuchsin
Basic fuchsin
What is also known as pararosaniline
Basic fuchsin
Ingredients in Schiff reagent
Basic fuchsin, HCL, sodium metabisulfite, DI
50% - 70% alcohol
Before processing, following fixation in Bouin solution tissue should be washed in:
The carbohydrate method useful for demonstrating amoeba is
Best carmine
This stain is used for demonstration of glycogen
Best carmine
What method will demo glycogen: Congo red Giemsa Mayer mucicarmine Best carmine
Best carmine
The problem shown in the image could have been prevented by
Better cleaning of forceps between specimens
Substitution of alcohol as the diluting solution for formaldehyde results in
Better preservation of glycogen
If malt diastase is used it is important to heat the solution
Beyond 40C, insufficient heat will not allow for complete digestion
Another stain that could be used to demo the bright structures in the image is
Bielschowski
The stain shown in the image is most likely
Bielschowski
A method that can be used to demo senile plaques is that of
Bielschowsky
What stain can aid in the diagnosis of Alzheimers
Bielschowsky
What stain is used for the demo of neurofibrillary tangles
Bielschowsky
What is the silver impregnation technique used to stain nervous tissue
Bielschowsky's Technique
The phenomenon shown in the image is
Birefringence
What information would you find on a tissue cassette?
Block number Year and surgical number Number of tissue pieces
Substances stained positive with the colloidal iron procedure will be
Blue
A deteriorating Schiff reagent will give a delayed reaction and show this color
Blue-Purple
The stain demoed in the image is most likely
Bodian
What stain demos axons
Bodian
What stain will demo a component of the peripheral nervous system
Bodian
List 2 methods for demoing nerve cell processes
Bodian, Holmes
The tissue shown in the image is
Bone marrow
When using the Bakers acid hematein method differentiation is accomplished by
Borax ferricyanide
Differentiator in Bakers acid hematein method
Borax ferrocyanide
What stain demonstrates DNA
Both Feulgen and the methyl green -pyronin
The technique shown in the image demonstrates
Both carboxylated and sulfated mucosubstances
Improper processing of tissue may result in
Both mushy sections or sections with chatter
what type of resin causes gradual fading of romanowsky stains over time
Both synthetic and natural
What fixative my lyse erythrocytes
Bouin
What is the fixative for: Masson Trichrome
Bouin
Which fixative contains picric acid, formalin, and acetic acid?
Bouin
What's the fixative for: connective tissue
Bouin Solution
The preferred fixative for the technique shown in the image is
Bouin solution
The feulgen reaction is unsatisfactory on tissue fixed in
Bouin soulution
Best secondary fixative for cytoplasmic (trichrome) staining
Bouin's
Holland's solution is a modification of
Bouins
The preferred fixative for the Masson Trichrome stain is
Bouins
Which two glycogen fixatives preserves the most glycogen?
Bouins & Rossman's
The tissue in the image is most likely
Brain
Which technique uses Iodine as a trapping agent?
Brown and Brenn's
What is the purpose of adding borax to working Methenamine Silver in Grocott's Methenamine Silver technique?
Buffer
The tissue in the image is from
CNS
Astrocytes are demoed by what procedure
Cajal
The stain most likely used in the image is
Cajal
What stain requires frozen sections
Cajal
What stain uses formalin ammonium bromide as the preferred fixative
Cajal
Glutaraldehyde
Cannot be used with Schiff's reagent, frequently used for fixation of specimens for EM, preserves ultrastructure, 2 hours or less for fixation
Acid mucopolysaccharides (an ionic heteroglycans) include
Carboxylated, sulfated&carboxylated, sulfated only
The problem shown in the image could have been prevented by
Carefully monitoring the endpoint of decalcification
These properties will be obscured if sections are overstained with either hematoxylin or metanil yellow
Carminophilic properties
Name a non-aqueous fixative
Carnoy
Acid fast stains may be negative if the tissue was fixed in
Carnoy Solution
Other fixatives used for best carmine
Carnoy and Bouin
Mayer mucicarmine stains these two types of mucins
Caroxylate and sulfated mucins
Another name for a basic or positively charged dye. ____________
Cationic dye
Hazards of osmium tetroxide
Causes toxic vapors, may fix corneas
Celestine Blue
Celestine blue, ferric ammonium sulfate, DI water...may be substituted for iron hematoxylin
The artifact seen in the image is
Cell shrinkage
Another excellent control for glycogen PAS reaction
Cervix
While the issue in the image can not be totally eliminated, it may be reduced by
Changing deparaffinization solutions often
Soaking a faced block can alleviate what common issue
Chatter on the sections
Another type of neutral polysaccharide (n-acetyl glucosamine containing:)
Chitin
Carbohydrates that don't require fixation
Chitin, starch, cellulose
Carnoy fluid is prepared with acetic acid, alcohol, and:
Chloroform
What clearing agents in neither flammable or combustible
Chloroform
Name the conjugated lipids which are hydrophobic
Cholesterol esters, mono ditriglycerides, waxes
A colored complex that includes a benzene ring and a chromophore. It is not considered a dye.
Chromagen
Oxidizing agent in Bauer-feulgen stain
Chromic acid
Is chromium pigment preventable, removable, and if so how would you remove it?
Chromium pigment is preventable and can be removed in 1% HCl in 70% alcohol for 30 minutes
What are the reagents in: Gridley
Chronic acid and Metanil Yellow
What are the reagents in: Grocott
Chronic and Periodic acid
Purge
Cleaning Cycles - Xylene - clean 100% ethanol - to remove xylene hot water - to remove buffer salts
Dehydrant, clearing, or infiltration/embedding: aliphatic hydrocarbons
Clearing
Dehydrant, clearing, or infiltration/embedding: benzene
Clearing
Dehydrant, clearing, or infiltration/embedding: cedarwood oil
Clearing
Dehydrant, clearing, or infiltration/embedding: chloroform
Clearing
Dehydrant, clearing, or infiltration/embedding: limonene derivatives
Clearing
Dehydrant, clearing, or infiltration/embedding: toluene
Clearing
Dehydrant, clearing, or infiltration/embedding: xylene
Clearing
List three connective tissues and a stain for each of them
Collagen: Masson trichrome Elastic: Verhoeff Reticulin: Gomori
Strongly sulfated mucins (CT and heparin sulfate) are stained by
Colloidal iron
What step is important for Alcian blue staining because some Alcianophilic structure hydrate slowly and if sections aren't fully this the result will be weak staining
Complete hydration of the sections during the deparaffinization and hydration steps
A Pre-treatment slide can be used to identify the difference between AL and AA
Congo Red
Amyloid can be demonstrated with
Congo red
Another stain that could be used to differentially demo the substance stained rose in the image is
Congo red
The staining technique shown in the image is most likely
Congo red
The technique shown in the image is most likely
Congo red
What stain shows green birefringence in amyloid deposits
Congo red
what stains are used to demo amyloid
Congo red and Thioflavin T
What stain is used for: amyloid
Congo red, crystal violet, thioflavin T
The cells wit the rose-violet cytoplasm shown in the image belong to what tissue type
Connective tissue
Hollande's Solution Ingredients
Copper Acetate, Picric Acid, Formaldehyde, Acetic Acid, DI Water (Modification of Bouin's, will decal small specimens of bone, used for biopsy specimens of the GI tract, Cupric Acetate stabilizes RBCs, must be washed before processing)
A stain applied after the main tissue component is highlighted. This serves as a contrast so the main component stands out better.
Counterstain
Overheating of the paraffin used for embedding may cause
Cracking of the block
Non-coagulant fixatives
Creates a gel that makes penetration by subsequent solutions difficult. Example: 10% NB Formalin Formaldehyde, Acetic acid
What stain will ID chromatolysis
Cresyl Echt Violet
What stain demos RER in neurons
Cresyl echt Violet
Nissl substance is demoed by
Cresyl echt violet
The rose staining in the image is due to what solution
Cresyl echt violet
Name a fungi well demoed by the colloidal iron stain
Cryptococcus neoformans
This organism is demonstrated with Mayer mucicarmine
Cryptococcus neoformans
In Gram's Stain technique what two solutions are combined together to form a High Molecular Weight Complex
Crystal Violet, Gram's Iodine
Another technique that could be used to demo the apple green colored substance in the image is
Crystal violet
In the Holzer technique, glial fibers are stained with
Crystal violet
The component stained red in the image is
DNA
The tissue component stained blue-green in the image is
DNA
The feulgen reaction demonstrates
DNA only
Without digestion acid mucins and sialomucins stain
Deep blue
The lines in the section shown are most likely due to
Defects in the blade edge
The dark areas in the image are due to
Deflected electrons
Dehydrant, clearing, or infiltration/embedding: ethyl alcohol
Dehydrant
Dehydrant, clearing, or infiltration/embedding: isopropanol
Dehydrant
Dehydrant, clearing, or infiltration/embedding: tetrahydrofuran
Dehydrant and clearing aka universal solvent
Dehydrant, clearing, or infiltration/embedding: dioxane
Dehydrant and clearing, aka universal solvent
A clearing agent for use in processing tissues for paraffin embedding must be miscible with the
Dehydrant and paraffin
The process of removing water form tissue is called
Dehydration
30% alcohol
Delicate tissues such as embryonic tissues may be distorted when placed in higher concentrations of alcohol, due to the diffusion currents that cross membranes and cause distortion so they should be started in concentrations of
Microscopic differentiation of tissue components in unstained tissue sections is difficult. Consequently, routine stains are used to:
Demonstrate the tissue components uniformly
Bielschowsky's Silver Technique demonatrates
Dendrites, axons, neurofibrils
Best knife to obtain section on EM
Diamond
These two enzymes act on glycogen to depolymerize it into smaller sugar units (maltose and glucose) that are washed out of the section
Diastase and alpha amylase
Name two substances that are used to demo specific carbohydrates
Diastase and hyaluronidase
What stain is used for: Glycogen
Diastase, periodic acid, Schiff reagent
The process of selectively removing excess or non-specific stain from tissue components where its presence is not desired. This may be accomplished by using solvents, acid or basic solutions, mordants, etc.
Differentiation
What is the function of: acid alcohol
Differentiation
What microtonal problem will most likely occur when sectioning the block shown
Difficulty forming ribbon
Name the substance that can be used to block the PAS reaction of many non-glycogen,PAS positive substance while preserving glycogen staining
Dimedone
Potassium Dichromate
Dissolves DNA, preserves lipids and mito,
The concentration of Alcian blue and the duration of staining may need to be varied with different
Dye lots
The principle of Oil Red O staining is based on
Dye solubility
Osmium Tetroxide
EM, Makes lipids insoluble
paraffin ribbons that fail to form may be the result of
Either too much or too little blade tilt
The type of microscopy shown in the image is
Electron
What type of microscope is focused by varying the strengths of magnetic fields
Electron microscope
The problem shown in the image could have been prevented by
Ensuring that both sections were embedded flat and at the same level
Eosin Solution
Eosin Y, 95% ethyl alcohol, GAA...must be acidic to develop a proper charge on proteins
Eosin Phloxine B
Eosin Y, Phloxine B,95% Ethanol, GAA
What does acid fuchsin stain in Masson's Trichrome technique?
Erythrosin
What are the types of conjugated lipids:
Ester lipids, phospholipids
Name a non additive fixative
Ethyl alcohol
microchatter
Excess heat during processing, especially in dehydration and clearing, can lead to removal of bound water and results:
When alcohol is used as the primary fixative one should expect
Excessive tissue shrinkage
See Schedule: Vacuum is used at all stations but heat is only used with the paraffin; specimens should have been fixed in NBF for at least 45 min before processing Formalin 10% neutral buffered 15 Min for all stations Alcohol 65% Alcohol 95% Alcohol 95% Absolute Alcohol Absolute Alcohol Xylene Xylene Paraffin Paraffin Paraffin What tissue would not fix well on the above schedule?
Excisional breast
The problem seen in the image could have been prevented by
Facing less aggressively
"The more the better" is a good practice for applying mounting medium
False
4-6 micrometer sections are recommended for crystal violet stains
False
Skin sections are embedded so that the epithelial surface is facedown in the block
False
T or F: 10% formalin is a 1:4 dilution of commercial formalin
False
T or F: A 3D image is obtained with the transmission electron microscope
False
T or F: A flotation bath is maintained at 37 C
False
T or F: A good fixative shoudl penetrate slowly
False
T or F: Acetic acid is a useful addition to many compound fixatives because of its shrinking action
False
T or F: Acid fast stains are satisfactory on tissue fixed in carnoy
False
T or F: B5 fixative contains formaldehyde and potassium dichromate
False
T or F: Bar codes may be linear or 3D
False
T or F: Basic dyes have a negative charge
False
T or F: Bouin solution contains picric acid, formaldehyde, and hydrochloric acid.
False
T or F: Chloroform is a universal solvent but cedarwood oil is not
False
T or F: Congo red and Thioflavin T prefer Orth fixation
False
T or F: Crooked paraffin ribbons are the result of either too much or too little blade tilt
False
T or F: Dust collecting on the cooling coils of the cryostat may cause the motor to run more slowly
False
T or F: Epoxy resins and glycol methacrylate are both safe when exposed to skin
False
T or F: Epoxy resins do not require the use of a transitional fluid
False
T or F: Fat is well preserved by Carnoy solution
False
T or F: Formalin ammonium bromide is a very good fixative for connective tissue
False
T or F: Formalin fixation stabilizes lipids
False
T or F: Functionally, the autonomic nervous system is under voluntary control
False
T or F: Gill hematoxylin is an iron hematoxylin
False
T or F: Gram+ organisms CANNOT be decolorized once stained with crystal violet
False
T or F: H pylori is a spirochete
False
T or F: Helly, Zenker, and Orth solutions all contain mercury
False
T or F: Hematein and hematin can be formed by combining with aluminum
False
T or F: Living cells are usually examined with the dark field microscope
False
T or F: Luxol fast blue is a water soluble dye
False
T or F: Mercury pigment can be removed by immersion in sodium thiosulfate
False
T or F: Nissl substance is composed of clumps of DNA
False
T or F: Paraffin and water soluble wax are acceptable for very hard tissues
False
T or F: Peanut oil is used in the Ziehl-Neelsen method
False
T or F: Prolonged washing of sections stained with PTAH will enhance the red staining of certain tissue components
False
T or F: Routine care of the microtome requires a thorough cleaning and oiling every 2 weeks
False
T or F: Since formalin is a coagulative fixative it is considered an excellent fixative for paraffin embedding
False
T or F: Steel blades are commonly used to section glycol methacrylate
False
T or F: Tetrahydrofuran will dehydrate tissue
False
T or F: The Holmes technique is an argentaffin method
False
T or F: The Schiff reaction may show false positives following chromate-containing fixatives
False
T or F: The angle formed by the block face and the cutting facet of the blade is known as the bevel angel
False
T or F: The fixing fluid considered best for the preservation of nuclear detail is buffered formalin
False
T or F: The giemsa stain will differentially stain the different types of bacteria
False
T or F: The temp of the room is very important when cutting frozen sections
False
T or F: The volume of the fixative should exceed the volume of the tissue by 1-2 times
False
T or F: When Luxol fast blue stain is combined with the Holmes silver nitrate stain, both glial fibers and myelin are demoed
False
T or F: Xylene is a universal solvent
False
T or F: Zenker fixative contains formaldehyde, mercuric chloride, and potassium dichromate
False
T or F: a chelating agent exchanges another ion for the calcium ion
False
T or F: alcian blue 2.5 is used in the demo of sulfated (only) mucosubstances
False
T or F: amyloid shows a yellow birefringence following staining with Congo red
False
T or F: benzene can be used to dehydrate tissue
False
T or F: bone sections should be embedded parallel to the long axis of the block
False
T or F: both formic and nitric acid are used to check the endpoint of decalcification
False
T or F: ethanol a clearing agent
False
T or F: glucose, sucrose, and other oligopolysaccarides can be demoed easily in tissue sections
False
T or F: glutataldehyde is one of the recommended fixatives for the PAS reaction
False
T or F: good Schiff reagent should be light pink
False
T or F: heating the solution is a good method of increasing the rate of decalcification
False
T or F: hematein and hematin can be formed by the action on formaldehyde
False
T or F: mercurial fixatives are satisfactory when stains for spirochetes are to be done
False
T or F: methyl green-pyronin and toluidine blue are used to demo fibroblasts
False
T or F: new instruments are validated by the manufacturer and may be out into use immediately
False
T or F: one result of incomplete clearing is poor infiltration with carbowax
False
T or F: paraffin sections with lengthwise splits are the result of either too little or too much blade tilt
False
T or F: specimens for electron microscopy are embedded in glycol methacrylate
False
T or F: the best method of checking zenker fixed tissue for the completeness of decalcification is radiologic examination
False
T or F: the routine alcian blue stain is done at a pH of 1.5
False
T or F: tissues are usually infiltrated with paraffin directly from an essential oil
False
T or F: toluene is the preferred clearing agent for microwave processing
False
T or F: viral organisms are easily demonstrated with special histochemical stains
False
T or F: when undecalcified bone is to be sectioned it must be embedded in carbowax
False
T or F:Resinous mounting media have an index of refraction much lower than that of the tissue
False
T or F:When examined microscopically 8 nm sections will show all nuclei in the same plane of focus
False
The nucleolus of plasma cells is stained green with the methyl green-pyronin technique
False
What kind of result would you get if you use tap water before adding Carbol Fuchsin on your slides when doing the Ziehl Neelson technique?
False Positive as there is mycobacteria in tap water
T or F: Diastase and hyaluronidase are used to demo amyloid
False, neither are used
T or F: Fluorescence microscopy is used to examine unstained cells while polarizing microscopy is not
False, neither are used
T or F: Alcian blue pH 1.0 and 2.5 both demonstrate glycogens
False, neither demo glycogen
T or F: Gomori reticulin prefers Zenker fixation but Periodic acid-methenamine silver does not
False, neither use Zenkers
T or F: Verhoeff and aldehyde fuchsin use acid differentiation
False, neither use acid differentiation
What control slide would you use when performing Oil Red O?
Fatty Liver cut at 10 microns
What are the unconjugated lipids
Fatty acid, cholesterol
What is the solution used in Gordon and Sweets (G&S) to cover aldehyde groups with Fe ions to prepare for silver binding to retic fibers?
Ferric Ammonium Sulphate or Iron Alum
Weigert's Hematoxylin
Ferric Chloride, DI water, HCl, hematoxylin, 95% alcohol...use no longer than 2-3 days
What chemical is used in the prep of Weigerts hematoxylin
Ferric chloride
What stain has a critical time for hydrolysis
Feulgen
List 4 connective tissue cells and suggest 1 stain for each
Fibroblasts: Not routinely demoed Mesenchymal: Not routinely demoed Adipose: Sudan Black Mast Cells: Toluidine Blue
The best stain for the demo of mycobacterium leprae is the
Fite
Methyl Alcohol
Fix touch preparations and blood smears
Routin processing
Fixation (10% NBF) Dehydration (70% Alcohol) (95%) (100%) Clearing (Xylene) Paraffin (Wax)
The first and most appropriate procedure in the preparation of a tissue for microscope examination is the choice of
Fixative
Problems associated with 95-100% Ethyl Alcohol
Flammable Poisonous Excessive hardness & shrinkage Government regulated
The problem in the image occurs during
Flotation
What type of microscope is used for the technique in the image
Fluorescent
Dioxane is a reagent that can be used
For both dehydrating and clearing tissues
The problem shown in the image is most likely due to
Forceps metastasis
After performing a PPB technique, you look under the microscope and can't find any evidence of ferritin on your control slide and patient slides. What could be the probable cause?
Forgot to add HCL to histochemical solution Potassium ferrocyanide was made up two months before use Tissue was fixed in Bouin's
routine surgical tissue processing example
Formal alcohol 2hrs 95% alcohol 2hr (2 changes total) Absolute alcohol 2 hrs(2 changes total) Xylene 2 hrs(2 changes total) Paraffin 3.5 hrs (4 changes total)
Fixative used in Bakers acid hematein method:
Formal calcium
Methylene bridges are formed during the reaction of certain tissue groups and
Formaldehyde
The reliability of the Schiff reagent May be checked by adding what to a small aliquot
Formaldehyde
10% Aqueous Formalin Ingredients
Formaldehyde and DI water (very hypotonic and may produce formalin pigment)
What is in: B-5
Formaldehyde and mercuric chloride
What is in: Orth
Formaldehyde and potassium dichromate
NH2
Formaldehyde primarily reacts by crosslinking this group.
Formalin Ammonium Bromide Ingredients
Formaldehyde, Ammonium Bromide, DI water (recommended only for CNS tissue, especially Cajal's astrocyte procedure-very acidic, lyses RBCs, and gives a direct positive Schiff reaction due to Fuelgen hydrolysis during fixation)
Calcium Formalin Ingredients
Formaldehyde, Calcium Chloride, DI water (recommended for fixation and preservation of phospholipids in tissue)
10% Neutralized Formalin Ingredients
Formaldehyde, DI water, Calcium or Magnesium Carbonate (solution becomes highly acidic after withdrawal from storage bottle)
Modified Millonig's Formalin Ingredients
Formaldehyde, DI water, Sodium Phosphate Monobasic, NaOH (Isotonic, pH 7.2-7.4, can be used for EM)
10% NBF Ingredients
Formaldehyde, Distilled Water, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic (pH 6.8, hypotonic)
Formalin Alcohol Ingredients
Formaldehyde, Ethyl Alcohol, DI Water (In addition to fixation, dehydration is also started)
10% Formalin Saline Ingredients
Formaldehyde, NaCl, DI water (isotonic, but may produce formalin pigment)
Acetate Formalin Ingredients
Formaldehyde, Sodium Acetate, DI water (can use calcium acetate and substitute for calcium formalin)
What is in: Helly
Formaldehyde, mercuric chloride, potassium dichromate
What is in: 10% NBF
Formaldehyde, sodium phosphate di and monobasic
The pigment seen in the image is most likely
Formalin
The preferred fixative for the technique shown in the image is
Formalin
A good fixative for central nervous tissue to be stained with silver or gold techniques is
Formalin ammonium bromide
Upon microscopic examination, an H & E stained section of routinely processed spleen shows small brown to black granules evenly distributed throughout the tissue. Suggest a possible causative agent and describe how this artifact can be removed.
Formalin pigment - Acid Formaldehyde Hematin (AFH) Formaldehyde / formalin, acid pH, hemoglobin (blood rich tissues) 1) Bring section to absolute alcohol 2) Place section in saturated picric acid for 5 min to 2 hours depending on the amount of pigment present 3) Wash for 15 to 20 min in running tap water 4) Proceed to desired stain
Is formalin pigment preventable, removable, and if so how would you remove it?
Formalin pigment is preventable, and can be removed with alcoholic picric acid or alkaline alcohol
additive fixatives (non "A" words)
Formalin, Mercury, Osmium, Glutaraldehyde
Non-coagulant Fixatives (For Good Girls Obey Their Parents Daily)-Jello (All non-coagulants are additive)
Formalin, glut, glyoxal, osmium tetroxide, porassium dichromate
For immunofluorescence the tissue should be
Fresh or unfixed
The best method for demonstration of lipids is
Frozen
Tissue for the procedure in the image must be
Frozen and unfixed
What's the fixative for: Enzyme histochemistry on muscle
Frozen, no fixation
What's the fixative for: immunofluorescence
Frozen, no fixation
A fast green counterstain May be used instead of hematoxylin when the stain is used to demonstrate
Fungi
What is stained by: Gridley
Fungi
What is stained by: PAS
Fungi
What is stained by: Grocott
Fungi and Pneumocystis jiroveci
Gold Chloride is a toning agent in which Connective Tissue staining technique
G&S
A medically important protozoan is
Giardia lamblia
The stain shown in the image is
Giemsa
name a hematoxylin stain that will stain the mucin in goblet cells
Gill
If this type of hematoxylin is used as the nuclear stain the mucin may have a bluish cast
Gill hematoxylin
Adjacent sections are stained with PAS, one with and the other without diastase digestion. A positive result on the slide without digestion and a negative result on the slide with digestion indicated the presence of
Glycogen
Diastase digestion increases the specificity for
Glycogen
PAS diastase digestion is a very sensitive histochemical method for
Glycogen
What would the result be for the different carbohydrates if you did a Alcian Blue technique on your slides.
Glycogen = Negative Neutral Mucin = Negative Acid Mucin = Positive
What would the result be for the different carbohydrates if you did a Southgates Mucicarmine technique on your slides.
Glycogen = Negative Neutral Mucin = Negative Acid Mucin = Positive
What would the result be for the different carbohydrates if you did a PAS Diastase technique on your slides.
Glycogen = Negative Neutral Mucin = Positive Acid Mucin = Negative
What would the result be for the different carbohydrates if you did a PAS technique on your slides.
Glycogen = Positive Neutral Mucin = Positive Acid Mucin = Negative
Results of best carmine stain
Glycogen = pink to red Nuclei = blue
Most commonly found carb in the body
Glycogen and mucin
Result of PAS w/distase
Glycogen will stain bright rose red on section labeled without and will be absent on slide labeled with
Neutral polysaccharides consist of: glucose containing (examples)
Glycogen, starch and cellulose
Epithelial mucins include
Glycoproteins
When using Alcian blue with hyaluronidase this type of mucin will not be affected and will be demonstrated even with digestion
Glycoproteins (epithelial mucins)
The tissue cells stained red in the image are
Goblet
Nonsulfated sialomucins are found in
Goblet cells, salivary glands and pancreas
Most silver stains use which of the following a a toning agent: sodium thiosulfate formaldehyde uranyl nitrate gold chloride
Gold chloride
The tissue shown in the image was most likely stained by what method
Gomori
Technique used to demonstrate both alpha and beta cells of the pancreatic islet of langerhorns
Gomori method
Name a stain that uses a separate formaldehyde reduction step
Gomori reticulin
What is the procedure for this reagent: Chromotrope 2R
Gomori trichrome
What is the procedure for this reagent: Aniline blue
Gomori trichrome, Masson trichrome
What is the procedure for this reagent: Diamine Silver
Gordon and Sweets
What is stained by: Brown and Brenn
Gram+ bacteria
A modification of the technique shown in the image stains collagen
Green
What stain will demo Pneumocystis jiroveci
Grocott
Name a hematoxylin where the hematein was traditionally formed by mercuric oxide
Harris
Which of the following contains aluminum ammonium sulfate?
Harris Hematoxylin
What is stained by: Steiner and Steiner
Heliobacter pylori, Treponema pallidum, and viral inclusions
What fixative contains formaldehyde
Helly
What fixative preserves erythrocytes in bone marrow sections
Helly
Which fixative contains formalin, potassium dichromate, and mercuric chloride?
Helly
What is more common in bloody tissue: Hematein or Hematin
Hematin
Ehrlich's Hematoxylin
Hematoxylin, 95% alcohol, DI Water, Glycerol, ammonium or potassium aluminum sulfate, GAA....air exposure for 2 weeks or add sodium iodate for immediate ripening, aluminum=mordant, used regressivley and progressively
Mayer's Hematoxylin
Hematoxylin, DI water, Sodium iodate, Ammonium or potassium aluminum sulfate, citric acid, chloral hydrate....aluminum=mordant, sodium iodate=oxidizer, citraic acid for pH, chloral hydrate prevents scum and ppts (used progressively)
Harris' Hematoxylin
Hematoxylin, absolute ethyl alcohol, ammonium aluminum sulfate, DI water, Mercuric Oxide..... mordant=aluminum, ripening agent=Mercuric Oxide (progressive and regressive)
Perl's Prussian Blue shows
Hemosiderin
When performing the Schiff staining method lipids are oxidized by
Performic acid and peracetic acid
Two oxidizing agents are used in the Schiff reaction for unsaturated lipids
Performic and peractic acid
What are the reagents in: PAS
Periodic Acid
What are the two oxidizing agents used in the PAS technique
Periodic Acid and Tap water
Kidney is the best control for
Periodic Acid-methenamine Silver stain
Has hydroxyl groups in the tissue
Perl's Prussian Blue
Name two secondary fluorescent methods used to detect lipids in formalin fixed frozen sections
Phosphate 3R, 3.4 Benzpyrene
What lipids are hydrophilic
Phospholipids, acid lycolipids, neutral glycolipids
Glial fibers are stained blue by
Phosphotungstic acid-hematoxylin
The technique shown in the image is most likely the
Phosphotungstic acid-hematoxylin
What is used to demo cross striations
Phosphotungstic acid-hematoxylin
What stains collagen reddish orange
Phosphotungstic acid-hematoxylin
What stains muscle blue
Phosphotungstic acid-hematoxylin
The technique shown in the image is what type of staining
Physical
Which fixative is the best in providing maximum preservation of glycogen in the tissue?
Picric Acid 95-100% Ethyl Alcohol
Bouin's Solution Ingredients
Picric Acid, Formaldehyde, Glacial Acetic Acid (Lyse RBCs, excellent for trichrome stain and for preserving structures with soft delicate textures, Fix less than 48hrs and transfer to alcohol, crisp nuclei)
Glycogen fixed in this will be more resistant to diastase digestion than with other fixatives
Picric acid
Why is the staining time critical in the Van Gieson reagent in the Verhoeff's Van Gieson technique?
Picric acid continues the differentiation of the elastic fibers
Discard Schiff reagent when solution turns
Pink
The issue shown in the image could have been prevented by
Placing the tissue in the fixative earlier
Results of the iodine method
Plant starch, amylose, amylopectin = blue, glycogen and amyloid = Red-brown
The technique used in the image utilizes a/an:
Polarizer and analyzer
To increase the specificity for amyloid Congo red stains should be examined by what type of microscopy
Polorizing
The tissue in the image has been screened for artificial pigment by the use of
Polorizing microscopy
PAS reaction use to demonstrate
Polysaccharides, neutral mucins and basement membranes
H&E stained slides of small intestine reveal muscle, collagen, and RBCs all stained the same shade of pink. This indicates
Poor differentiation of eosin
Orth's Solution Ingredients
Potassium Dichromate, Sodium Sulfate, DI Water, Formaldehyde (Not good general purpose fix, preferred foe demo of chromaffin granules in the adrenal medulla to diagnose pheochromocytoma)
Select the oxidizer in the Gordon and Sweets silver impregnation procedure for reticulin fibers
Potassium Permanganate
Mercuric Chloride
Powerful protein coagulant, enhances staining by leaving tissue receptive to dyes, produces pigment that can be removed with iodine and sodium thiosulfate
Acetone
Precipitant, non additive fixative, used for demo of enzymes especially acid and alkaline phosphatase, done at refrigerator temp and dehydration simultaneous, fixative for brain tissue when stains for rabies are needed
Ethyl Alcohol
Preserve water soluble tissue components, ie Urate crystals in Gout and glycogen, preserves pigments, dissolves fat, overhardens tissue
Paraffin is cooled as rapidly as possible after embedding tissue in order to
Prevent the formation of large crystals
Why is mercuric chloride avoided for glycogen Staining
Produces an uneven glycogen staining
Nuclear staining with Alcian may occur if the stain is
Prolonged
Depending on the reagent used what could cause the tissue to become over hardened?
Prolonged fixation
An example of a solvent used in the oil Red O and Sudan black B methods that prevents the loss of lipids during fat staining is
Propylene glycol
Zinc (Sulfate)
Protein Coagulant, nuclear detail, better paraffin infiltration
Zinc salts are added to some formalin fixatives to
Provide superior nuclear detail
The end product of the technique shown in the image is
Prussian blue
In the Gridley procedure, the aldehyde fuchsin stain will attach to
Schiff reagent
In the PAS reaction this reagent is cancer causing
Schiff reagent
See Schedule: Vacuum is used at all stations but heat is only used with the paraffin; specimens should have been fixed in NBF for at least 45 min before processing Formalin 10% neutral buffered 15 Min for all stations Alcohol 65% Alcohol 95% Alcohol 95% Absolute Alcohol Absolute Alcohol Xylene Xylene Paraffin Paraffin Paraffin If the power went off 2 hours and 20 min after beginning processing, the tissue would be in what station
Second paraffin
The tissue section in the image is a
Section of kidney
One advantage of the paraffin technique is that
Serial sections are easy to obtain
The tissue shown in the block
Should have been centered in the mold
While performing Gordon and Sweet's technique, you notice that your tissue has fallen off the slide. What is the reason for this?
Silver is a very alkaline solution and has the tendacy to cause the tissue to fall of the slide
The impregnating solution used in the technique shown in the image contains
Silver protein
After performing Grocott's Methenamine Silver you notice that the tissue is green but you can not find Fungus on the slide. What could be the probable cause?
Silver was not made correctly There was no Fungus on the control slide to begin with Tissues were left in silver solution at room temperature for 30 min.
List three types of muscle fibers
Skeletal, cardiac, smooth
After completing Verhoeff's Van Gieson technique you notice on your control slide that your background seems muddy. What is the reason for this?
Slides in the sodium thiosulphate for too long
The problem shown in the image is most likely caused by
Slow freezing
What tissue is shown in the image
Small intestine
The problem in the image could have been helped by
Soaking the faces block
What chemical changes hematoxylin to heamtein
Sodium iodate
What chemicals are used in the prep of Mayer hematoxylin
Sodium iodate and ammonium aluminum sulfate
The tissue shown in the image is
Spinal cord
The major source of the issue in the image is tissue carryover in
Staining
The following are properties of mucin (4)
Staining with basic dyes, metachromatic, precipitated by acetic acid (except gastric mucin), soluble in alkaline solution
Due to the anhydrous aluminum chloride reacting with atmospheric moisture and water to give of vapors of hydrogen chloride of this solution it should be used in the hood
Stock mucicarmine solution
This solution will gradually deteriorate even with refrigeration so solution over several months old should be discarded
Stock mucicarmine solutions
Zenker and Helly's Solution Ingredients
Stock: Mercuric Chloride, Potassium Dichromate, Sodium Sulfate, DI Water Zenkers: Stock and GAA (Lyse Erythrocytes, good nuclear fixative, fix and decal needle biopsy specimens of bone marrow, dissolves iron, good for Mallory's PTAH) Hellys': Stock and Formaldehyde (Preserve Erythrocytes) (Post treat for mercury, not good for silver staining, cannot be stored indefinitely)
Tissue is fixed in order to:
Stop autolysis
Picric Acid
Strong coagulant of nucleoprotein, leaves DNA soluble, recommended for glycogen fixation, leaves tissue receptive to acid dyes
Another technique that could be used to demo the substance stained red in the image is the
Sudan Black B
The alcian blue stain performed at a pH of 0 demos
Sulfated acid mucopolysaccarides
Results of Alcian blue 1.0
Sulfated mucosubstances = pale blue Background = pink to red Nuclei = Red
This type of medium is used with lipids
Synthetic resin
unfixed frozen sections
The BEST method of preparing tissue for enzyme demonstration. In order to prevent a decrease in enzymatic activity, processing that requires heat is avoided.
Tissue fixed in glutaraldehyde is not satisfactory for
The PAS reaction
A pathologist is reading the gross description report and it indicates that the patient sample they are examining should be uterus but under the microscope they see tissue from a breast on the slide. What could be the reason for the pathologist not receiving the correct specimen with the gross description report? (3)
The Pathologist Assistant opened two specimen containers at a time and grabbed the wrong specimen while grossing. -The Pathologist Assistant gave the wrong requisition to the wrong specimen after grossing. -The Technologist while embedding opened two cassettes at one time and put the wrong tissue in the wax mold with the wrong cassette on top.
The arrow in the top left of the image is pointing to
The basement membrane
The tissue component that should be present but is missing in the image is
The epithelium
what stain demonstrates RNA
The methyl green-pyronin
granular deposition of the silver is seen on the control stained with the Gordon and Sweets reticulin method. One possible explanation is that
The reagents may have been old
decalcification
The removal of calcium deposits, essential for good embedding procedure. specimen should be cut 3-4mm for adequate penetration. over use of decalcification acids can cause nuclear basophilia. Occurs with simple acids, chelation and ion exchange Methods used to check for completeness include chemical, mechanical and radiographic.
Control slides for the technique shown in the image may give poor results if
The slides have been cut and stored for a long time
After completion of a Gordon and Sweet's technique, the reticulum is demonstrated as pale grey fibers. Which of the following would be the possible cause?
The time in the Ferric Ammonium Sulphate was too short
See Schedule: Vacuum is used at all stations but heat is only used with the paraffin; specimens should have been fixed in NBF for at least 45 min before processing Formalin 10% neutral buffered 15 Min for all stations Alcohol 65% Alcohol 95% Alcohol 95% Absolute Alcohol Absolute Alcohol Xylene Xylene Paraffin Paraffin Paraffin If the power went off 2 hours and 20 min after beginning processing and remained off for 1 hour, what should happen to the tissue
The tissue should be embedded
An H&E slide shows reddish brown stained nuclei, pink cytoplasm, and bright rose-red erythrocytes. these results indicate
The use of over ripened hematoxylin
For periodic acid, these type of sections may require longer oxidation time
Thinner sections(1-3)
The stain used in the image is most likely
Thioflavin S
A fluorescent used for the demo of amyloid is
Thioflavin T
Another technique that might be used to demonstrate the substance stained red in the image is
Thioflavin T
The dye used for the technique demoed in the image is most likely
Thioflavin T
what stain is primarily a fluorescence technique
Thioflavin T
The problem shown in the image could most likely be corrected by
Tightening the block and blade clamps
What is the most likely cause of eosin being too pale in an H+E stain?
Tissue not rinsed well enough in water after blueing agent used
What will happen to a tissue if acetic acid is used as a fixative alone?
Tissue will swell
Why is it necessary to oxidize Hematoxylin before using it as a staining solution?
To convert it to hematein, a dye
30% sucrose
To maintain tissue morphology of fixed tissue that is to be frozen, the tissue should be infiltrated with ___
The primary purpose of fixation is
To stop enzymatic action
The stain shown in the image is most likely
Toluidine blue
What stain demos mast cells metachromatically
Toluidine blue
Name the two metachromatic dyes that stain mast cells
Toluidine blue and aldehyde fuchsin
What is the function of: gold chloride
Toning
The problem in the image could have been from
Too large a clearance angle
One cause of the problem in the image is
Too much blade tilt
A disadvantage of the use of Dioxane in processing tissues is that it is
Toxic
T or F: A fixative stops autolysis and putrefaction
True
T or F: A good Grocott methenamine silver stain shoes organisms with a crisp black cell wall and a visible internal structure
True
T or F: A neuron only has 1 axon
True
T or F: A problem may result from allowing the slides to dry during Gram staining process
True
T or F: Acetic acid is an excellent nuclear fixative
True
T or F: Acetone is sometimes used when a rapid acting fixative is needed
True
T or F: An increase in temperature usually increases the rate of staining
True
T or F: An increase or decrease in the pH of the staining solution can alter staining by changing tissue and/or dye charges
True
T or F: Aniline oil used in the Holzer technique is very toxic and rated by NIOSH as neoplastic
True
T or F: Another name for the ocular of a microscope is the eye piece
True
T or F: Any fixative containing mercuric salts will leave a pigment in the tissue
True
T or F: Both Xylene and Tetrahydrofuran will clear tissue for infilatration
True
T or F: Both mercury pigment and acid hematin are deposited in tissue during formalin fixation
True
T or F: Cedarwood oil will make the tissue transparent but chloroform will not.
True
T or F: Chrome pigment can be prevented by washing the tissue with water following fixation
True
T or F: Concentrated commercial solutions of formaldehyde are 37-40% by weight of the gas formaldehyde dissolved in water
True
T or F: Copper is used in the Bodian impregnating solution to increase the differentiation between neural and connective tissues
True
T or F: Eosin is differentiated by dehydrating alcohols
True
T or F: Ependymal cells are epithelial cells
True
T or F: Epoxy resins do not tolerate water
True
T or F: Excess Bouin should be removed before processing
True
T or F: Fixation in Helly solution will preserve erythrocytes, while fixation in Zenker solution will not
True
T or F: Formalin penetrates rapidly but fixes slowly
True
T or F: Formalin pigment can be removed from tissue by immersion in alcoholic picric acid
True
T or F: Frozen sections can be stained with toluidine blue O
True
T or F: Gluteraldehyde is frequently used to fix specimens for electron mycroscopy
True
T or F: Glyoxal fixatives are rapid acting compared to formaldehyde
True
T or F: Gomori reticulin and periodic acid-methenamine silver both require an oxidation step
True
T or F: H pylori is readily demoed by a Romanowski type stain
True
T or F: Heat and desiccation are forms of fixation
True
T or F: Household microwave instruments may not be used in the laboratory
True
T or F: Iodine serves as a mordant in the Gram stain
True
T or F: Kinyoun and Auramine-rhodamine both contain phenol
True
T or F: Kinyoun and Auramine-rhodamine both demonstrate mycobacteria
True
T or F: Methyl green-pyronin and Toluidine blue are used to demo connective tissue cells
True
T or F: Orth solution is the best fixative for pheochromocytomas when IHC is not to be performed
True
T or F: Osmium tetroxide chemically fixes fat
True
T or F: Oxidizers are sometimes used for differentiation
True
T or F: Resinous mounting media are usually dissolved in toluene, xylene, or xylene substitutes
True
T or F: Rod shapes bacteria are called bacilli
True
T or F: Side by side comparisons are the best form of instrument validation
True
T or F: Stains for nissl substance can be used to ID the presence of neurons in tumor tissue
True
T or F: The Griffey stain is more intense than the PAS
True
T or F: The analyzer of the polarizing microscope is paced between the specimen and th eye
True
T or F: The mordant is applied after the primary due in the Gram stain
True
T or F: The stains for spirochetes are argyrophil techniques
True
T or F: Tissue left in a fixative beyond the defined amount of time may become excessively hard
True
T or F: Water in the flotation bath is a possible source of contamination on sections to be stained for microorganisms
True
T or F: When used as a counterstain for Luxol Fast Blue, cresyl echt violet must be used in an acid solution
True
T or F: Xylene and Tetrahydrofuran are both toxic
True
T or F: Xylene and toluene are aromatic hydrocarbons
True
T or F: Xylene will not mix with water
True
T or F: Zinc is a satisfactory replacement for mercury in some fixative solutions
True
T or F: auramine-rhodamine is a fluorescent technique
True
T or F: both formic and nitric acid are used to decalcify specimens
True
T or F: carbowax is a blend of liquid polyethylene glycol and wax
True
T or F: ferric chloride is both a mordant and oxidizer
True
T or F: formic acid is used with ion exchange resins
True
T or F: frozen sections are specified for processing tissue for the demonstration of most enzymes
True
T or F: glycogen containing tissue fixed with Bouins solution may show resistance to diastase digestion
True
T or F: hematein can be formed by the actions of air or light
True
T or F: hyaluronidase is used to digest some connective tissue mucins
True
T or F: if phosphate buffered formalin fixation is followed by dehydration beginning with 80% alcohol, phosphates may be precipitated
True
T or F: if the hist tech is skilled, then the most critical factor in the lab becomes the care and maintenance of lab instruments
True
T or F: if tissues have been fixed in an aqueous fixative, uric acid crystals can not be demonstrated
True
T or F: ion exchange resin and electrolyte methods of decalcification fall under the broad heading of "acid methods"
True
T or F: methylene blue can be used in the processing alcohols to dye tissues for identification when embedding
True
T or F: myelin is responsible for the color of white matter
True
T or F: nitric acid frequently impairs staining reactions
True
T or F: nitric acid may cause tissue damage after 48 hours
True
T or F: one cause of uneven staining of the H&E may be water contamination of the clearing agent on the processor
True
T or F: overheating the paraffin used for infiltration will cause shrinkage and over hardening if the tissue
True
T or F: paraffin will dissolve fat out during processing
True
T or F: sections processed by the cellodin method show less shrinkage than when processed by the paraffin method
True
T or F: the end product in the colloidal iron method is Prussian blue
True
T or F: tissue containing H pylori is a satisfactory control for the Diff-Quik Giemsa modification
True
T or F: tissue will contain ice crystal artifacts when frozen slowly
True
T or F:Slow tissue of freezing leads to the formation of ice crystals
True
T or F:the addition of acid to the crystal violet staining solution reduces background staining
True
The control tissue for Mayer mucicarmine should be
Unautolyzed colon, small intestine or appendix
Glycol methacrylate is commonly used on what type of tissue
Undecalcified bone
After performing Gram's stain you noticed that nuclei are red, tissue is yellow, gram positive bacteria are purple and gram negative bacteria are purple. What is the reason for this result?
Underdecolorized in the Acetone (Acetone step in between Gram's Iodine and Basic
Reagents that both clear and dehydrate are called
Universal solvents
Absolute alcohol is indicated as a primary fixative if the tissue is to be processed for the demonstration of
Urate crystals
After completing Masson's Trichrome technique, you notice your control slide doesn't have nuclei stained. What is the reason for this?
Used Aluminum hematoxylin to stain nuclei
The technique shown in the image
Uses a fluorescent ragged antibody
What is not a possible cause of the artifact in the image
Using a dull blade
The issue in the image often occurs when sectioning what type of tissue
Uterus
In what technique does Ferric Chloride act as an oxidizing agent, differentiating agent, and mordant
VVG
The issue in the image will most likely produce a slide with what artifact
Venetian blind atrifact
What stain uses ferric chloride as a mordant
Verhoeff
What is the procedure for this reagent: Iodine
Verhoeff, primary stain
The technique shown in the image is the
Verhoeff-van Gieson
Which of the following methods best demos elastic tissue: Verhoeff-van Gieson Silver impregnation Gomori Trichrome PAS
Verhoeff-van Gieson
Name a glycogen storage disease demonstrated by best carmine
Von Gierke
Cloudiness will result when the slides are placed in the alcohols (during Alcian blue 2.5) if the slides aren't
Washed well with water after the nuclear fast Red stain
Fixatives containing chromate salts usually require
Washing in water
The dehydration and clearing steps can be omitted when using
Water soluble wax
Results of Alcian blue 2.5
Weakly acidic sulfated mucins, hyaluronic acid, sialomucins = dark blue Background = pink to red Nuclei= blue
a. Disposal of used 10% NBF
Wear appropriate PPE - mask, gloves and safety glasses. Work in a chemical fume hood. Mix with formalex in a ratio of 1:5 with formalin. Leave for a minimum of 1 hour (when a precipitate has formed, the fixative has been "neutralized"). This solution can now safely be poured down the sink with running tap water.
Name a hematoxylin that is not an aluminum hematoxylin
Weigerts
Which hematoxylin solution must be used within a few days after preparation
Weigerts
name a hematoxylin that is not readily decolorized with acidic solutions
Weigerts
Macroscopic evaluation can be used to judge the quality of which technique
Weil
Spinal cord is an excellent control for which technique
Weil
The technique shown in the image is most likely
Weil
List 2 techniques for demoing myelin
Weil and Luxol fast blue
List two regressive methods of staining nerve components
Weil and Luxol fast blue
-20C
When tissue is frozen slowly at this temperature large artifactual holes are often seen as a result of large ice crystals.
Generally an increase in the temperature of the fixative solution
Will increase the speed of fixation
osmium tetroxide
Will interfere with staining. Cell cytoplasm has little affinity for anionic/acid dye such as eosin but will readily accept cationic (basic dies).when used as a secondary fixative it should be used under a chemical hood because it readily vaporizes and will fix nasal mucosa or conjunctiva of eye. Specimens for EM can not be left in this for more than 2-4hr. immuno EM can not be done.
Precipitation of the dye on sections which commonly occurs during staining with best carmine is the result of evaporation of the ammonia in the carmine solution so this step should be done to avoid this
Working solution should be filters and used in a closed container
A light microscope has a maximum useful magnification of
X1000
The high dry objective on most microscopes has a magnification of aprox
X45
What is used in clearing portion of prosessor ?
Xylene
When used for clearing, cedarwood oil must be followed by
Xylene
Limonene
Xylene substitute that causes sensitization and allergic reactions in some individuals with prolonged use.
Problems associated with Picric Acid
Yellow discolouration of tissue Hemolyzes RBCs Dissolves Iron Explosive when dry
What's the fixative for: electron microscopy
Zambini and osmium tetroxide
What fixative contains acetic acid
Zenker
What fixatives must be washed out with running water
Zenker and Helly
What two fixatives contain mercuric chloride and potassium dichromate
Zenker and Helly
What's the fixative for: phosphotungstic acid hematoxylin
Zenker solution
The X ray test to determine the endpoint not used on tissue fixed in
Zenker's - mercury will intrfere
Tissue must be washed in water after fixation in
Zenkers
Goagulants (ZAPAM)
Zinc, Alcohol, Picric acid, Acetone, Mercury
H&E stained sections show a brown microcrystalline deposit lying on top of the tissue, it is especially heavy in the bloody areas of the tissue. This pigment can most likely be removed by treating the tissue with
alcoholic picric acid
Silver impregnation stains for reticulin depend on the formation of which of the following chemical groups: quinoid aldehyde carboxyl amino
aldehyde
Another method for demoing the tissue component stained black in the image is
aldehyde fuchsin
The technique demoed in the image is the
aldehyde fuchsin
The technique shown in the image depends upon the presence of
aldehydes
When used as a primary fixative for electron microscopy, Millonig formaldehyde
allows both light and electron microscopy
cytocentrifugation
allows essentially all the cells in a sparsely cellular fluid specimen to be deposited onto a slide, while the fluid is absorbed away onto a filter paper.
The substance demoed in the image could be removed by
alpha amylase
hematoxylin is converted to a dye lake in the Mayer formula by adding what to the solution
ammonium aluminum sulfate
The diamine silver complex is formed by a reaction between silver and
ammonium hydroxide
Congo red demonstrates
amyloid
The substance stained red in the image is
amyloid
The substance stained blue in the image is
an acid mucopolysaccaride
What type of solution should be used to calibrate the pH meter to allow for adjustments to the pH of the Warthin-Starry stain
an acidic solution
Tissue is processed with the following reagents: 10% neutral-buffered formalin 2 changes 65 % alcohol 1 change 95% alcohol 2 changes 100% alcohol 2 changes aliphatic hydrocarbon 2 changes paraffin 3 changes The tissue does not seem to be well cleared and infiltrated.This problem might be solved by adding:
an aliphatic hydrocarbon and deleting one of the 95% alcohols
An electron gun is used with
an electron microscope
The stain shown in the image has stained reticulin in
an incorrect granular pattern
An auxochrome is
an ionizing group present in dyes
The problem in the image can be corrected by removing the coverslip
and carefully applying a new one
The problem shown in the image could be corrected best by removing the cover slip
and decolorizing and restaining the section
The problem in the image could best be corrected by removing the cover slip
and redehydrating and reclearing with fresh solutions
Into which dye category does eosin fall
anionic
Control slide for Gomori's Aldehyde Fuchsin
aorta or skin
Control slide for Verhoeff's Van Gieson
aorta or skin
When a lens for a light microscope has been corrected for three colors it is said to be
apochromatic
The tissue has loosened from the chuck while frozen sectioning. This can most likely be prevented in the future by
applying the embedding medium to a warm chuck
a light microscope has a resolution of
approx 0,2 nm
antigens
are altered by high temperatures and repeated exposure to heated paraffin during reprocessing may alter the epitope sites such that false negative results are seen
natural resins are rarely used for mounting sections today because they
are inherently acidic
essential oils
are used as clearing agents. when used it is necessary to remove the oil with a hydrocarbon such as xylene prior to paraffin infiltration.
aldehyde fixatives
are used for EM because they preserve cell ultrastructure. They must be followed by secondary osmium tetroxide fixation to preserve lipids.
urate crystals
are water soluble and must be fixed with an aqueous based fixative such as absolute alcohol.
Stains for the demo of spirochetes are based on their property of
argyrophilia
Boiun solution functions in the Masson Trichrome stain
as a mordant
Tetrahydrofuran
as a universal solvent, causes diffusion currents that can harm delicate tissue morphology in small specimens such as lung biopsy.
A feulgen stain is required on a section of Bouin fixed lymph node. the best course of action is to
ask if there is tissue in another fixative
The scanning objective on the light microscope is found
at the lower end of the body tube
Which fungal stain is a fluorescence technique
auramine-rhodamine
The structure stained black in the image is
axons
Brown & Brenn gram stain demonstrates
bacteria
The protocol for a new automated stainer is needed, and one thing that must be decided is how often to change solutions. the best choice is to rotate solutions
based on slides stained
The tissue components stained black in the image is
basement membrane
Carbol-fuchsin contains
basic fuchsin and phenol
Gomori aldehyde fuchsin solution contains
basic fuchsin, hydrochloric acid, and paraldehyde
A tissue component that takes up a cationic dye is said to be
basophilic
vimentin IHC
best antibody for determining when tissue has been overfixed
One advantage of primary aldehyde fixation is that
better penetration is obtained than with osmium
A microscope that has two eyepieces is
binocular
Calcium in the Von Kossa technique
black - silver impregnation
Fungi in the Grocott's Methenamine Silver technique
black - silver impregnation
Hemosiderin after a Perl's Prussian Blue
blue
Streptococcus after Ziehl Neelsen technique
blue - colour of background
Nuclei in the Congo Red technique
blue - stained with aluminum hematoxylin
In the Brown-Hopps modification of the Gram stain for tissues, Gram+ organisms appear
blue-black
The tissue shown in the image is
bone marrow
The technique shown in the image demos
both carboxylated and sulfated mucosubstances
Tissue to be stained by the procedure demonstrated in the image should not be fixed in
bouins
A specimen will not be considered optimally fixed if the nuclei show
bubbling
Van Kossa shows
calcium
chromate pigment
can be prevented by treating tissue with running water prior to processing.
superior nuclear details
can be seen when comparing zinc-formalin to formalin.
What can be added to an aqueous mounting medium to prevent bleeding of aniline dyes into the surrounding medium
cane sugar
The component of basement membranes that is usually demoed with special stains is
carbohydrate
Both gomori reticulin and periodic acid-methenamine silver stains are based on
carbohydrate demonstration
The PAS reaction will demonstrate fungi, because the cell wall contains
carbohydrates
The technique in the image depends on the presence of
carbohydrates
bis-chloromethyl ether
carcinogen that can be chemically formed by a reaction between formaldehyde and hydrochloric acid.
fatty cell membrane
cause many aqueous fixatives to penetrate poorly
Xylene or toluene must be used after
cedarwood oil
A dye that may be substituted for hematoxylin in routine staining is
celestine blue
The differential staining achieved with the Gram stain is due to differences in the bacterial
cell wall
Control slide for Luxol Fast Blue
cerebellum
The nervous tissue in the image is
cerebellum
coagulant fixatives
change the sponge work of proteins into a mesh-like network that allows solutions to readily penetrate or gain entry into the interior of the tissue. Includes zinc salts, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, acetone and picric acid.
While staining a rack of slides, it is notes that the water following the rehydrating alcohols turns very cloudy. the can most likely be corrected by
changing the alcohols
Giemsa staining is poor on a bone marrow section. This problem might be corrected in the future by
changing the pH of the staining solution
Glutaraldehyde
dialdehyde that will give nonspecific PAS staining. Penetrates slow/poorly. Most frequently used for EM because it preserves ultrastructure. Tends to overharden tissue so its normally used at 2-4%. Breaks down when exposed to oxygen. Irreversibly blocks tissue antigen. for EM follow fixation with phosphate buffer wash and postfixation in osmium tetroxide
The technique shown in the image requires the use of
diamine silver
Name a substance that digests glycogen
diastase
The problem in the image could be prevented by
differentiating the eosin better in the lower dehydrating alcohols
The most important step in regressive hematoxylin staining is
differentiation in acid-alcohol
Water soluble waxes will infiltrate the tissue
directly from aqueous fixatives
indicators for poor fixation of EM specimens
disrupted mitochondrial membranes
water-soluble waxes
do not require dehydration and clearing.
Carbowax processing (Polyethylene glycol)
does not require the use of solvents that would dissolve lipids so the fat remains in tissue following infiltration with this. Has a major disadvantage of being water soluble, and therefore sections cannot be floated on a water bath.
Verhoeff's Van Gieson
elastic
Gomori's Aldehyde Fuchsin demonstrates
elastic and mast cells
The component stained purple in the image is
elastic fibers
The tissue component stained black in the image is
elastic fibers
The Verhoeff-van Gieson shows both orange collagen and muscle. This most likely could be corrected in the future by
ensuring that the picric acid solution is saturated
Control sections stained with Congo red show only yellow and no green birefringence. This could probably be corrected in the future by
ensuring that the sections are cut at 8-10 nanometers
The problem shown in the image is
eosin does not show three shades
Southgate's mucicarmine
epithelial acid mucins
The cells showing the intense red cytoplasm in the image are
erythrocytes
one possible cause for the problem in the image is
evaporation of the mounting medium
The Verhoeff method differentiates with
excess mordant
Oxidation or ripening of hematoxylin can be done by
exposure to atmospheric oxygen, or using sodium iodate, mercuric oxide, or potassium permanganate (Oxidized hematox has little affinity for tissue, but becomes strong when combined with a metallic mordant)
Various size holes are noted in sections of paraffin embedded liver on the water bath. With further ribboning, these holes decrease and disappear. The most likely cause of the holes is
facing the block too aggressivlely
T of F: Mercury pigments and acid hematin are water soluble
false
T or F: A colorless background indicated a problem with the cresyl echt violet stain
false
T or F: Acid and basic fuchsin are used in the Gordon and Sweets stain
false
T or F: An over heated cryostat motor is frequently cause by ice buildup in the cryostat chamber
false
T or F: Bouin solution is a good fixative for tissue to be stained with the Fuelgen reaction
false
T or F: Congo red and Thioflavin T are best set on 4-5 nanometer sections
false
T or F: If one wishes to prevent the formation of a pigment, formalin solutions must be buffered to a pH of >7.0
false
T or F: In the central nervous system, myelin is made by the astrocytes
false
T or F: Masson trichome and Phosphotungstic acid-hematoxylin are used to demo elastic tissue
false
T or F: Poly-L-lysine is a common additive to the flotation bath
false
T or F: Sodium iodate and ammonium aluminum sulfate are used to adjust the pH of hematoxylin
false
T or F:Sodium iodate and ammonium aluminum sulfate are used to make Weigerts hematoxylin
false
T or F: the primary stain for Verhoeff and aldehyde fuchsin are stable for months
false, neither are stable
T or F: Masson trichrome and van Gieson stains are used to demo elastic tissue
false, neither are used
T or F: Adipose and mast cells fix best on Carnoy solution
false, neither tissue prefers carnoy
Control slide for Oil Red O
fatty liver
A chemical used in the technique shown in the image for both oxidation and differentiation is
ferric chloride
what chemical ripens hematoxylin solutions
ferric chloride
A blue-black precipitate is seen on an H&E stain slide This could probably be prevented in the future by
filtering the hematoxylin
Marked, non-specific, background staining is noted on a section stained with the PAS technique. This could be the result of
fixation with glutaraldehyde
steps to processing
fixation, dehydration, clearing and infiltration.
additive fixative
fixative that contains mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, zinc sulfate or chloride. These chemically link or add themselves to the tissue and change it with this action.
aldehydes
fixatives that contain these mask antigenic sites and hamper IHC localization of antigens
Electron microscope studies on a section of tumor fixed in 10% NBF reveal very poor cell preservation. This can be prevented in the future by
fixing some of the tumor on gluteraldehyde solution
Tissues are subjected to a series of different reagents in a closed processor by
fluid transfer
The type of microscopy used in the image is
fluorescence
Mercuric oxide was used in the original formula for Harris hematoxylin to
form hematein
Both B-5 and Orth contain
formaldehyde
The reducing agent in most reticulin stains is
formaldehyde
The preferred fixative for sections to be stained by the cajal technique is
formalin ammonium bromide
Methylen bridges
formation is reversible using the tap water
The technique shown int he the image depends on the
formation of aldehydes
The problem in the image most likely could be corrected in the future by using mounting medium that is
from a freshly opened bottle
The oil red O stain requires which of the following sections: paraffin cellodin frozen plastic
frozen
The sections used for the technique shown in the image must be
frozen
The sections for the stain shown in the image must be
frozen and cut at 20-30 micrometers
The staining technique shown in the image is the
fuelgen
Glycol methacrylate
functions as an infiltrating medium that is converted to a solid
The term mycosis is used to describe a disease caused by
fungi
Grocott's Methenamine Silver demonstrates
fungi (eg. Pneumocystis)
See Schedule: Formalin, 10% 2 hrs (no heat, vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) 95 % alcohol 1 hr (only vacuum) 95 % Alcohol 45 min (no heat, no vacuum) Absolute Alcohol 45 min (only vacuum) Absolute Alcohol 1 hr (no heat, no vacuum) Xylene 1 hr no heat, no vacuum) Xylene 1 hr (only vacuum) Paraffin 30 min (no vacuum) Paraffin 1 hr (no vacuum) Paraffin 1.5 hr (vacuum) What type of tissue would not fix well on this schedule
gastric biopsies
name a good example of a polychrome stain
geimsa
Hematoxylin & Eosin shows
general stain
Aliphatic hydrocarbons
generally called alkanes. Xylene substitute that is not always compatible with all mounting media. They are lower in toxicity and sensitization than aromatic hydrocarbons used as clearing agents.
Artificial precipitate seen in the Grocott stain may be the result of using
glassware that was not chemically cleaned
Steps for Fixation for EM with Glutaraldehyde
glute>Phosphate buffer>sucrose solution>Osmium Tetroxide
The substance demoed in the image is
glycogen
The technique shown in the image is the best stain for the demonstration of
glycogen
Everything in the image is incorrectly stained except
goblet cells
neuron cell bodies are found primarily in
gray matter of the CNS
Control slide for Bielschowsky's Silver Technique
grey matter
Control slide for Cresyl Echt Violet
grey matter
Cytoplasm if underdifferentiated in the VVG technique
grey to black
resins (epon and Spurr)
harden by polymerization. repeat exposure to these may cause dermatitis
toluene
has low viscosity and will clear tissues at 20C. higherviscosity clearants will clear more slower at 20C than lower viscosity clearants.
Depolymerization of paraformaldehyde occurs with
heating the solution
Why specimens are separated by different types of tissue?
helps to elliminate mistakes
The active staining chemical in ripened hematoxylin solutions is
hematein
What is formed when hematoxylin is subjected to the action if sodium iodate
hematein
The problem in the image could probably be corrected int he future by decreasing the time in
hematoxylin
hetero-chromatin is stained by
hematoxylin
List the reagents contained in the staining solution used in the weil method
hematoxylin, ferric ammonium sulfate
Name a substance that removes connective tissue mucin
hyaluronidase
The tissue stained blue in the image would show decreased or absent staining if treated with
hyaluronidase
paraffin wax
hydrocarbons produced by cracking of petrolium, kept 2-4 * above melting point
picric acid
hydrolyze nuclei causes shrinkage
The developer in the Warthin-Starry stain is
hydroquinine
The basic structure of filamentous fungi is the
hypha
acid
in order to stop the decalcification process of this solution tissue must be washed in water. Carbon dioxide by product is formed. Should be neutralized with 1% sodiumbicarbonate before flushing down the drain
Multiplem small pieces must be puted
ina row or located centrally
At microtomy, formalin fixed and paraffin embedded kidney sections are soft and mushy. This is most likely due to
inadequate dehydration and clearing
The problem shown in the image was most likely caused by
inadequate differentiation
H&E stained sections of liver show very dark nuclei and some blue staining of the cytoplasm. this is most likely caused by
inadequate differentiation of the hematoxylin
No staining of the glomerular basement membrane can be seen microscopically on a control section of kidney. This may be the result of
inadequate oxidation
microscopic examination of a section stained with the Gomori reticulin stain and counterstained with nuclear fast red shows cloudiness of both the section and the slide. This is most likely the result of
inadequate washing after nuclear fast red
Well stained sections show blue-black white matter and brown gray matter. This indicates that the sections were
inadequately treated with borax-ferricyanide
The problem in the image is due to
incomplete dehydration of the section
one possible cause of the problem in the image is
incomplete drying of the slides before staining
H&E stained sections of liver in 10% NBF show a marked difference in staining between the periphery and the center of the tissue. More nuclear bubbling is also noted in the center of the section. This is most likely due to
incomplete fixation prior to beginning dehydration
One possible cause of the problem in the image is
incomplete removal of paraffin
Vacuum
increases the rate of infiltration of processing fluids, thus decreasing the time necessary to complete the steps in processing.
During cryotomy, sections of varying thickness are obtained. This can most likely be corrected by
increasing the blade tilt
Control slide for Brown & Brenn gram stain
infected tissue
The nuclear counterstain most likely used in the technique in the image is
iron hematoxylin
The primary staining solution used in the technique in the image is
iron hematoxylin
elastic fibers are demoed in the method shown in the image by
iron hematoxylin
During microtomy, the sections lift from the blade as the block is raised, the most likely cause
is a dull blade
Glycol methacrylate (GMA)
is an embedding medium that will tolerate a small amount of water after processing. Recommended for very thin sections for light microscopy evaluation.
Paraffin wax
is an inert mixture of long chain hydrocarbons produced from petroleum processing. 3 changes are recommended during processsing with this.
nuclear bubbling
is caused by incomplete fixation.
The last dehydrating absolute alcohol in the H&E staining setup is very pink. this indicates that the alcohol
is contaminated with water
95% alcohol
is equal to 80% isopropyl alcohol and 100%methyl alcohol
Paraffin that is considered soft
is most useful when thick sections are desired
primary chromate fixative
is necessary for the preservation of chromaffin granules.
Chloroform
is not flammable or combustible, but when heated, it may form a toxic gas.
hematoxylin only becomes a dye when it
is oxidized
isopentane (prechilled to -150C)
is the best freezing method for muscle enzyme demonstartion
benzene
is very volatile and thus rarely used as a clearing agent, but one advantage when it is use is that it evaporates rapidly from paraffin at its melting point, so waxes don't need to be changed on processor.
If cells are placed in a hypertonic solution containing what could happen?
it aill shrink
Ammonium ion exchange
keeps acid free from calcium ions.
The epithelium in the image is
keratinizing stratified squamous
Control slide for Masson's Trichrome
kidney
The tissue shown in the image is
kidney
Control slide for Ziehl Neelsen
known TB lung
Control slide for Congo Red
known amyloid slide
Control slide for Grocott's Methenamine Silver
known fungi slide
Control slide for Perl's Prussian Blue
known hemosiderosis slide
Direct Method (IHC Staining Method)
labeled Ab of known specificity is used to identify Ags in the patient's tissue
Formalin fixed tissue shows very faded blue staining with the Masson trichrome technique. The most likely explanation is that the sections were
left too long in the final acetic solution
Romanowsky type stains are typically preferred for the demonstration of
leukocytes
Neutral mucins in a PAS technique if potassium permanganate is used as the oxidizing agent
light pink - colour of background - CHO overoxidized from aldehydes to acid state
Mordants are used to
link tissue constituents more closely to the dye
One advantage of primary osmium tetroxide fixation is that
lipids are rendered insoluble
The oil red O stain might be used to demo
liposarcomas
A good control for reticulin stains is
liver
Control slide for Periodic Acid Schiff's(PAS) glycogen
liver
Another technique that can be used to demo the substance stained black in the image is
luxol Fast Blue
what stain begins its differentiation with an alkaline solution
luxol Fast blue
B-5 fixative is recommended for what tissue type
lymph nodes
Fungi in the Periodic Acid Schiff (PAS) technique
magenta - chitin binds with Schiffs
Acetic acid is added to Harris hematoxylin to
make nuclear staining more specific
harder paraffin
makes cutting decalcified sections of bone easy
Water bubbles are seen microscopically on an H&E slide. This could probably be prevented in the future by
making sure the dehydration step is complete
A stain that might be used to demo cirrhosis of the liver is the
masson Trichrome
What is considered a connective tissue stain
masson Trichrome
What is the procedure for this reagent: Beibrich scarlet
masson trichrome
Toluidine blue is used to demo which of the following cells: plasma mast fibroblasts macrphages
mast
Toluidine Blue Gomori's Aldehyde Fuchsin Giemsa All techniques demonstrates?
mast cells
Geimsa stain demonstrates
mast cells or H. pylori
Toluidine blue stain shows
mast cells or H. pylory
underdecalcified tissue
may be soaked in 5% HCL to remove calcium fromthe surface of the block
electrolytic methods of decal
may cause heat damage to the specimen because of the heat generated by the method.
Name a hematoxylin where the pH was traditionally adjusted by the addition of citric acid
mayer
To differentiate Cryptococcus neoformans from other yeast like fungi, which stain should be performed
mayer mucicarmine
Gomori Burtner demonstrates
melanin
Neither 10% NBF nor Bouin contain
mercuric chloride
B-5 Fixative
mercuric chloride sodium acetate (anhydrous) distilled water formaldehyde-should be added just use tissue can not stay in this solution indefinitely.recommended for subsequent AFB staining
Coagulant Fixatives (My Cat Eats Meat And Potatoes Au gratin)-sponge network
mercuric chloride, ethanol, methanol, acetone, picric acid
Additive Fixatives (My Cat Picked At Frank's Good Table)-adds something
mercuric chloride, picric acid, formaldehyde, glutaraldehyde, osmium tetroxide
Fluorescence microscopy requires the use of what type of lamp
mercury or halogen
What pigment can be removed with Lugol iodine
mercury pigment
Is mercury pigment preventable, removable, and if so how would you remove it?
mercury pigment is not preventable, and can be removed with iodine followed by Sodium Thiosulfate
The rose-red staining shown in the image is
metachromatic
The technique in the image uses
methenamine silver
Plasma cells can be demoed with
methyl green-pyronin
The stain used in the image is
methyl green-pyronin
What stain demonstrates the RER
methyl green-pyronin
What stain turns plasma cell cytoplasm red
methyl green-pyronin
What stains the cytoplasm of plasma cells rose
methyl green-pyronin
Tissue components can be measured with the light microscope in a process known as
micrometry
Good fixation is indicated on an electron micrograph of a section of a plasma cell if the
mitochondria show no swelling or disruption
Hollande solution
modified version of Bouin solution copper acetate -stabilizes RBC picric acid -hydrolyze nuclei formaldehyde acetic acid -lysis RBC distilled water can decalcify small specimens of bone
Incomplete infiltration
moist block , soft smells of xylene mushy Correction - 2-3 changes of parrafin wax
To link hematoxylin to tissue DNA what must be added
mordant
The higher the plastic point
more support
The problem in the image is
mounting medium on top of cover slip
Microscopic examination of an H&E section of kidney proves difficult. Some areas of the tissue can not be brought into focus, while others show excellent detail. this is most likely due to
mounting medium on top of the cover glass
A decrease in section transparency can be caused by using
mounting medium that has become too thick
Another technique that is frequently used to aid in the ID of the organisms stained blue in the image is
mucicarmine
The technique shown in the image is most likely
mucicarmine
The difference between cryptococcus neoformans and other yeast like fungi is that only C. neoformans has a capsule containing
mucin
The substance stained red in the image is
mucin
Quenching (snap freezing)
muscle biopsy , uses clamps to prevent muscle from contraction , detales will not seen
Pap smears
must be placed in 95% alcohol for 15min before staining or nuclei will appear foggy and lack detail
The carbol-fuchsin methods are specific for
mycobacteria
The auramine-rhodamine technique will demo
mycobacterium tuberculosis
Luxol fast blue stain shows
myelin
The material stained blue in the image is
myelin
The tissue component stained blue-black in the image is
myelin
What is the tissue component is demoed by: Luxol Fast Blue
myelin
The structures stained blue in the image are
myelin sheaths
For the most transparency and clarity when viewing well stained microscopic sections, the refractive index of the mounting medium should be
near that of the tissue
What type of resin dissolves in water
neither natural nor synthetic
A neuron is a
nerve cell
The structures stained black in the image are
nerve fibers
What is the tissue component is demoed by: Protargol
nerve fibers
Nissl substance is present in
neurons
Oil Red O technique shows
neutral fats (simple fats)
Periodic Acid Schiff's shows
neutral mucin, glycogen
If a ribbon splits while cutting paraffin sections, the trouble is most likely due to
nicks in the blade edge
Carboxylated mucins after Alcian Blue technique ph 1.0
no demonstration - not blue
The microwave oven created heat on staining solutions by
non ionizing radiation
Fine elastic fibres in Verhoeff's Van Gieson (VVG) after leaving in Van Gieson reagent too long
not black/not demonstrated - iron hematoxylin removed by the acid
Reticulin fibres if iron alum is skipped in the Gordon and Sweet's
not demonstrated
Melanin if potassium permanganate was used as an oxidizing agent in the Gomori Burtner technique
not demonstrated - removed
Neutral lipids for Oil Red O cut from wax block
not demonstrated - removed
Harris hematoxylin is used on tissue sections to stain
nuclei
ultrastructural preservation
obtained at physiologic pH 7.2-7.4
Incomplete dehydration
of tissues during processing will cause poor staining and lack of nuclear detail after H&E staining.
The stain shown in the image probably results from
old sensitizing reagent
warthin-starry technique
only use formlin fixation
nonadditive fixatives
organic compounds such as acetone and the alcohols. These act on tissue without chemically combining to it. example: alcohols precipitate or coagulate protein but do not add to the tissue.
Lipids are soluble in
organic solvents
What is the fixative for: Paraffin processed fat
osmium tetroxide
What must be used under a hood because it readily vaporizes and will fix nasal mucosa?
osmium tetroxide
fat is chemicaly fixed and maintained in tissue by
osmium tetroxide
See Schedule: Formalin, 10% 2 hrs (no heat, vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) 95 % alcohol 1 hr (only vacuum) 95 % Alcohol 45 min (no heat, no vacuum) Absolute Alcohol 45 min (only vacuum) Absolute Alcohol 1 hr (no heat, no vacuum) Xylene 1 hr no heat, no vacuum) Xylene 1 hr (only vacuum) Paraffin 30 min (no vacuum) Paraffin 1 hr (no vacuum) Paraffin 1.5 hr (vacuum) If the above schedule began at 5 pm, and the power cut out at 11:45 pm, and remained off for 2 hrs, the tissue would be
overhardened
Sections of small intestine show orange goblet cells that are partially obscured by the yellow background. This is most likely the result of
overstaining with metanil yellow
Bodian stained sections show light gray nerve fibers. One possible explanation is that the sections were left too long in
oxalic acid
Ripening of hematoxylin is a process of
oxidation
The first step in most reticulin methods is
oxidation
When used in a silver stain for reticulin, phosphomolybdic acid functions as the
oxidizer
The PAS stain differs from the Gridley technique in the
oxidizer used
Mercuric oxide in H+E is used as a/an?
oxidizing agent
One possible cause of the problem in the image is
pH is too high
Glycogen, after diastase in the Periodic Acid Schiff technique
pale pink - background colour
The tissue in the image is
pancreas
Specimens embedded in what type of material are floated on a warm water bath
paraffin
H&E stained slides show uneven staining, with some areas well stained and others unstained. These results indicate that most likely the
paraffin was not completely removed
The pure polymer of formaldehyde is know as
paraformaldehyde
Zamboni solution (buffered picric acid formaldehyde/PAF)
paraformaldehyde (heat to 60C to dissociate ) picric acid, saturated aqueous (double filtered) add 2.52% aqueous sodium hydroxide dropwise (to alkalinize) allow to cool then add 1000mL phosphate buffer final pH should be 7.3. Allows secondary fixation with osmium tetroxide. Good for both light and EM. stable at room temp
When the magnification can be changed without the need to refocus, the microscope objectives are said to be
parfocal
A disadvantage of osmium tetroxide fixation is that osmium
penetrates poorly
Mercuric chloride
penetrates poorly and causes excessive shrinkage. Mercuric salts will produce a brownish-black pigment in tissue.
phosphate buffered formaldehyde is used in the first 2 stations of a closed processor. A white precipitate is forming in the processor tubing, and the tissue is more difficult to cut than usual. One of the first things to check to correct t he problem is the
percent alcohol used as the first dehydrant
In the Hotchkiss-McManus modification of the PAS technique, aldehydes are formed by
periodic acid
The oxidizer used in the technique shown in the image is
periodic acid
The tissue in the image is
peripheral nerve
A Masson stain has been requested and the supply of phosphomolybdic acid has been depleted. The best action is to substitute
phosphotungstic acid
In the Masson Trichrome, Biebrich scarlet is removed from collagen by
phosphotungstic acid
The solution responsible for staining of the muscle in the image contains
picric acid
Bouin solution
picric acid (saturated aqueous solution)- hydrolyze nuclei formaldehyde acetic acid, glacial -lysis RBC frequently used for GI biopsys and has a mordanting effect on tissue. Tissue fixed in this should be washed and stored in 70% alcohol
The components on van Gieson solution are
picric acid and acid fuchsin
Blocked tissue, which has been fixed in Bouin solution, is pulled from the file after being stored for several years. new sections are cut and stained with H&E, no nuclear staining is noted on the new sections although the original sections stained very well. The most likely explanation is
picric acid was not sufficiently removed before processing
Melanin in the Luxol fast blue if overdifferentiated with lithium carbonate
pink
Cytoplasm in an H&E if over differentiated with acid alcohol
pink - only affects nuclei
The cells that show intense pink cytoplasmic staining in the image are most likely
plasma cells
For Gomory Burtner Technique use which forceps?
plastic
Doubly refractile particles are examined using
polarized light
Birefringent substances are best examined with what type of microscope
polarizing
What type of microscopy can help ID crystals
polarizing
What type of microscopy is demoed in the image
polarizing
Mallory PTAH stain is
polychromatic
The technique shown in the image is
polychromatic
During H&E staining if ammonia is incompletely removed by washing the result may be
poor staining with eosin
Orth fixative contains
potassium dichromate
Mallory PTAH solution is ripened for immediate use by
potassium permanganate
nucleic acids
preferred fixatives are acetic alcohol and carnoy solution
The problem shown in the image could most likely be corrected by
preping fresh colloidal iron solution
Synthetic resins are preferred over natural resins because synthetic resins
preserve the intensity of the stains with time
Fat droplets are seen in the tissue spaces of an oil red o stained section. This most likely resulted from
pressing on the cover glass to remove air bubbles
Ethylene glycol functions in Gill hematoxylin solutions to
prevent a surface sheen
infiltration
process of saturating tissue with the medium that will be used for embedding. The coomplete process depends on complete replacement of clearing agent from the tissue, and the time necessary for that process will be increased in thick and/or dense tissues; time necessary will be decreased for thin and/or less dense tissue samples. Heat should only be applied to this step, as heating other processing reagents will generally result in hard and brittle tissues.
Mast cells in a Gomori's Aldehyde Fuchsin technique
purple - GAF not specific for elastic
The reagent that gives the rose -red color int he section in the image is
pyronin
H&E stained sections fixed in formalin show a brown microcrystalline deposit lying on top of the tissue; this can most likely be prevented in the future by
raising the pH of the formalin to > 6.0
Michel solution
reccomend transport medium for tissue undergoing immunofluorescence studies or needing to be unfixed for a long transportation.
acetone
recommended for frozen sections of brain tissue to be stained for a diagnosis of rabies. can be used to fix tissue for the demonstration of cell surface antigens. over harden tissue but can preserve glycogen and some enzymes
Calcium Formalin (Calcium Chloride)
recommended for the fixation and preservation of phospholipids
Formalin ammonium bromide
recommended for the fixation of tissue for staining with Cajal method for astrocyte demonstration
HER2 testing
recommended that tissue be fixed for no less than 6hrs, no more than 48
1mm cubed
recommended tissue sections to be fixed in osmium tetroxide for EM.
Epithelial acid mucins in the Southgate's mucicarmine technique
red
Gram positive bacteria if over-differentiated after crystal violet
red
In the Fite method, the organisms stain
red
Van Gieson solution stains collagen
red
With both the Masson and Gomori trichrome stains, muscle stains
red
Mycobacterium after Ziehl Neelsen technique
red - AFB stain with carbol fuchsin
Nuclei after Masson's trichrome using an aluminum Hematoxylin
red - same as cytoplasm
Neutral lipids for Oil Red O cut on a cryostat
red to orange
Formaldehyde is used in the technique in the image as a
reducer
The technique shown in the image is
regressive
dehydration
removal of water. Dioxane (universal solvent) , methanol and ethanol can all be used. When tissue is not completely fixed before this step fixation in alcohol occurs and leads to increased eosinophilia at the center of the tissue.
After Schiff reagent, tissues are rinsed in a sulfite solution to
remove the excess leucofuchsin
Serial sectioning
requires all sections be saved on sequently nubered slides
PTAH staining
requires mordanting in Zenker or Bouin if tissue was fixed in formalin. Identifies rhabdomyosarcoma
When used as a primary fixative for electron microscopy, gluteraldehyde
requires that specimens be removed after 2-4 hours
The ability of the microscope to separate small details is defined as
resolution
Gordon & Sweet's demonstrates
reticulin
The technique shown in the image demos
reticulin
The tissue component stained black in the image is
reticulin
The connective tissue has failed to stain yellow with the Movat pentachrome stain. One possible cause is that the
safran solution contains water
These stains may stain nuclei but are more frequently used as counter stains
safranin, nuclear-fast red, methylene blue, thionin, toluidine blue O, gallein
Electron micrographs of buffered picric acid formaldehyde (PAF, Zamboni's) fixation showed marked extraction of the lipids. This indicated that most likely
secondary osmium tetroxide fixation was not used.
Sections stained with the Luxol fast blue-holmes silver nitrate technique show very pale blue myelin and gray axons. The most likely explanation is that the
sections were cut too thin
The bright structures seen in the image are most likely due to
senile plaques
When ferric ammonium sulfate is used on a silver stain for reticulin, it functions as the
sensitizer
Aqueous mounting media have an index of refraction that is
significantly below that of the tissue
The blue stained tissue component in the image is
skeletal muscle
Control slide for Gomori Burtner
skin
The tissue in the image is
skin
The tissue shown in the image is
skin
A large quantity of delafield hematoxylin stock solution must be maintained because of
slow hematein formation
A good control for the technique in the image is
small intestine
Control slide for Alcian Blue
small intestine
Control slide for Hematoxylin & Eosin
small intestine
Control slide for PAS for neutral mucins
small intestine
glyoxal
smallest dialdehyde. rapid fixation can occur between 4-6hrs. good for most special stains including PAS despite it being a dialdehyde. Not good for IHCs, or identification of calcium in breast. Best when used at pH4.
The red stained circular tissue component in the image is
smooth muscle
Hematein is formed in Mayer hematoxylin solution by the addition of
sodium iodate
A black precipitate is noted on a periodic acid-methenamine silver stained section that has been on the pathologists desk for about a month. The stain did not have any precipitate originally. This indicates that most likely the
sodium thiosulfate step may have been omitted
carbohydrates
some are lost during fixation and with many fixatives the retention of glycogen is thought to result from entrapment by the fixed proteins.
acetic acid
sometimes added to fixatives to reduce shrinkage (causes swelling), marked lysis of erythrocyte and coagulate/precipitation of nucleoproteins are characteristics of fixatives containing:
Most critical step of embedding
specimen orientation
Biopsy
specimens are small and process well in the time and solutions utilized in microwave processing.
One advantage of buffered picric acid-formaldehyde fixation (PAF, Zamboni's) is that
specimens may remain in the fixative indefinitely
Control slide for Gordon & Sweet's
spleen or liver
What is the reason for using glycerol in H+E?
stabiliser
fats
stains for these are done of frozen sections fixed in neutral buffered formalin.will be lost in routine processing
calcium carbonate
storing stock solutions of formalin with this can cause the stock to become acidic.
Iron hematoxylin, rather than aluminum hematoxylin is usually used to satin nuclei in the trichrome procedures because
subsequent staining solutions are acidic
Cracks around tissue in the block is caused by
super cooling of the cold plate
What type of resin dries and becomes non-sticky very quickly
synthetic
Fixation with NBF will cause tissue cytoplasm to
take up more hematoxylin
During microtomy, it is noted that most of the tissue is very hard and shrunken. One of the first things to check to prevent its happening in the future is the:
temperature of the infiltrating paraffin
factors affecting fixation
temperature, size, volume ratio (15 to 20) and time
Demonstrates large and small elastic fibers easily
the Gomori Aldehyde Fuchsin technique
plastic paraffin
the additions of these to paraffin increase hardness and support for hard and dense tissue.
The component or structure stained red in the image is
the basement membrane
70% alcohol
the best solution for long term storage of tissue; this preserves both routine and immunohistochemical staining properties.
putrefaction
the breakdown of tissue by bacterial action
DNA can be demonstrated with
the feulgen reaction
10% neutral buffered formalin
the fixative of choice if a specimen will not be processed for several days
Collagen capsules should be
the last part to hit the knife
Care must be taken when using automatic coverslippers to ensure that
the mounting medium applied is correct for long term storeage
osmium tetroxide and chromic acid
the only 2 chemicals that can fix lipids so that they are not lost in subsequent processing steps.
The solution of buffered 4% paraformaldehyde is cloudy. The mos likely explanation is that the
the paraformaldehyde is not completely depolymerized
Indirect Method (IHC Staining Method)
the patient's serum is added to tissue sections containing known Ags to test the patient for the presence of Abs to those Ags. Also means a method of using 2 Abs to detect Ag in a patient's tissue
clearing
the processing step that removes alcohol from tissues prior to infiltration with wax. determining factors for a good agent include; cost, removal by paraffin and flammability
Sections stained with PTAH show blue glial fibers, but the neurons are unstained. One possible cause is that
the sections were washed too long before clearing and mounting
Microscopic review of H&E stained slides reveal an artifact known as 'cornflaking'. This is caused by
the slide drying before mounting
electrolytic decalcification
the specimen to be decalcified is attached to the anode. The reason for this is that calcium ions will migrate to the cathode. Positively-charged calcium ions are attracted to the negatively-charged cathode.
glycogen
the storage form of glucose (blood sugar). ethyl alcohol is the fixative of choice.
68C
the temperature of the microwave oven should not exceed
the problem in the image could be caused by
the use of a warped cover slip
H&E stained slides show hazy blue nuclei, but recuts from the tissue processed a week previously and stained in the same basket show excellent nuclear staining. One possible cause is
the use of too much heat during processing
Compressed or wrinkled sections may be caused by
the wrong blade tilt
Incomplete clearing
tissue -opaque, cloudy, soft, shrinks Correction: Xylene to remove wax 2 changes of xylene to remove alcohol reimpregnation wax
70C
tissue antigens will be denatured by exposure to paraffin at ____, rendering them unable to be demonstrated
The problem in the image is
tissue drying before coverslipping
potassium dichromate
tissue fixed in solutions containing this will be very receptive to eosin staining.
enzyme histochemical studies
tissue should be treated with 30% sucrose and 1% gum acacia and can be stored at 4C for several weeks for:
epoxy resins
tissue to be infiltrated and embedded in these must be completely dehydrated prior embedding
Extended sitting of tissue in melted paraffin cause
tissue to shrink and harden
Embedding of small intestine
tissue wall on edge - all layers are visible
paraffin sections that are compressed, wrinkled, or jammed are the result of
too little blade tilt
paraffin sections that display microscopic chatter are the result of
too much blade tilt
At the time of embedding, a white deposit is noted on tissue fixed in unbuffered zinc formalin and then transferred to phosphate buffered formalin. One possible explanation could be that the tissue was
transferred to buffered formalin without washing
List the two most common types on electron microscopy
transmission and scanning
The goblet cells in a section of small intestine fail to show any blue staining with the Movat pentachrome stain. This could possibly be the result of
treating the slides with alkaline alcohol too briefly
T or F: Dehydration of formalin fixed specimens should begin with 65 -70% alcohol
true
T or F: Gray matter is practically colorless in a correctly performed Luxol fast blue stain
true
T or F: Hematin is an artifact
true
T or F: Masson trichrome and van Gieson are used to differentiate smooth muscle from collagen fibers
true
T or F: Most tissue contamination from floaters occurs in the deparaffination steps
true
T or F: Some staining kits may have the problem of disproportionate solution volumes
true
T or F: Staining can be influenced by the fixative used
true
T or F: The Weil technique uses both excess mordant and an oxidizer for the differentiation steps
true
T or F: Verhoeff and aldehyde fuchsin are elastic stains
true
T or F: both a cryostat and a flotation baht must be kept free of debris
true
T or F: paraffin sections will not adhere well to clean, untreated, and uncharged glass slides
true
T or F: peripheral nerve is good control for the Bodian stain
true
Sections 90 nm thick are commonly cut with an
ultramicrotome
Mushy sections can be caused by
under dehydration
dioxane
used as a universal solvent, which indicates that it is miscible with water, alcohols, hydrocarbons, and paraffins. Can be used for both clearing and dehydration
butanol (butyl alcohol)
used for dehydrating plant material
microwave processing
uses isopropyl alcohol for both dehydration and clearing becuase it is miscible with paraffin wax
Bodian stained sections show marked precipitation on the sections. This could probably be prevented in the future by
using chemically cleaned glassware
The Masson trichrome stain shows only a faint grayish pink staining of the muscle. This could most likely be prevented in the future by
using fresh acid fuchsin-biebrich scarlet solution
Black precipitate is seen on sections stained with the Gomori method for reticulin. This is most likely the result of
using glassware that was not chemically clean
What stain uses a saturated solution of picric acid in the primary stain
van Gieson
What stains collagen red
van Gieson
Cytoplasm in an H&E if the blueing agent is not properly removed
very pale pink
Nissl substance using the Cresyl echt violet technique
violet
Mast cells using Geimsa or Toluidine Blue
violet - both metachromatic dyes
H&E stained slides reveal brown pigment like stippling and rare glossy black nuclei, this most likely has been caused by a mounting medium that
was applied after letting the slide dry
H & E stained sections show very uneven staining of the tissue, with poor nuclear detail. One possible cause is:
water in the clearing agent
Hard and shrunken
when infiltrating paraffin becomes too hot (2-4C over the melting point) tissue becomes
incomplete fixation
will cause cracks in the tissue and smudgy nuclei.
low viscosity dehydrants
will dehydrate more rapidly at 20°C than higher viscosity dehydrants.
Zinc-formalin
will give poor ultrastructure preservation.
10% ammonium (sodium or potassium) hydroxide in 70% alcohol
will remove formalin pigment if slides are placed in solution for 30min- 3 hours before staining. When pH falls below 6 formalin pigment can form.
iodine-sodium thiosulfate
will remove the mercury pigment caused by mercury containing fixatives
When would you need to use a halogen lamp
with a florescence light microscope
Incomplete dehydration , corrective actions
xylene alcohol (several changes) xylene Paraffin
clearing agents
xylene, toluene, benzene, chloroform, acetone, essential oils, limonene reagents (xylene substitutes) and aliphatic hydrocarbons. Must be miscible with the reagents used directly before (dehydrants) and after(infiltrating media).
Nuclei in the Brown and Brenn Gram stain if over differentiated with picric acid/acetone
yellow
Nuclei in Gomori's Aldehyde Fuchsin technique
yellow - no specific stain for nuclei
Which fixative is considered a substitute for B5?
zinc formalin
Water soluble wax processing
• -contact with water must be avoided in sectioning • -there must be minimum distortion of tissue • -fat stains can be done on the sections • -procedure may require several hours
Tissues embedded in glycol mrthacrylate are commonly cut with
a rotary microtome
The black stained structure in the center of the image is
a senile plaque
Differentiating in the H&E stain is an example of using
a weak acid
chromaffin granules
demonstration of these is used for the diagnosis of pheochromocytoma.
Verhoeff's Elastic Stain
-demo pathologic changes in elastic fibers -regressive method, tissue is overstained by a soluble lake of hematoxylin-ferric chloride-iodine, -ferric chloride (diff) and iodine serve as mordants, and have an oxidizing function that assists in converting hematoxylin to hematein -Fixative: any well fixed tissue, paraffin 4-5um, QC-section of aorta embedded on edge or a cross sxn of a large artery -Reagents:10% Ferric Chloride, Verhoeff's elastic stain (Hematoxylin, ferric chloride, Lugol's iodine), Van Gieson's Solution (acid fuchsin, picric acid), Sodium Thiosulfate -Results: Elastic fibers- blue to blue/black, nuclei-blue to black, collagen-red, other tissue elements-yellow
Gordon and Sweets Stain for Reticular Fibers
-demo reticular fibers to differentiate and diagnose certain types of tumors or liver diseases -Fix 10% NBF, paraffin 4-5um, QC-liver -Reagents: Ammoniacal Silver Soln (Silver Nitrate, NaOH)-impregnate, Potassium Permanganate Soln (oxidize), Oxalic Acid (bleach), Ferric Ammonium Sulfate (sensitize), Formalin (reduce), Gold Chloride (tone), Sodium Thiosulfate (remove unreacted silver), Nuclear Fast Red -Results: reticulin-black (less background and nuclear staining)
Gomori's Stain for Reticular Fibers
-demo reticular fibers to differentiate and diagnose certain types of tumors or liver diseases -Fix: 10% NBF, paraffin 4-5um, QC-liver -Reagents: Ammoniacal Silver Solution (Silver Nitrate Solution, Potassium Hydroxide)-impregnate, Potassium Permanganate Soln (oxidizer), Potassium Metabisulfite Soln (Diff), Ferric Ammonium Sulfate Soln (Sensitize), Formalin (reducer), Gold Chloride Soln (toner), Sodium thiosulfuate Soln (remove unreacted silver-fix), Nuclear Fast Red -Results: Reticulin-black, Collagen-taupe
Warthin Starry Technique for Spirochetes
-demo spirochetes -argyrophyl method-need chemical reducer -Fix 10%NBF, paraffin 4-5um, QC-tissue w/ spirochetes -Reagents: Citric acid, Developer (silver nitrate crystals, acidulated water, gelatin soln, hydroquinone) 1% silver nitrate (impregnate) -Results: Spirochetes-black, background-pale yellow to light brown
Dieterle Method for Spirochetes and Legionella Orgs
-demo spirochetes or causative orgs of legionnellosis -Fix:10%NBF, paraffin 4-5um, QC-tissue containing spirochetes and legionella orgs -Reagents: Alcoholic Uranyl Nitrate, Silver Nitrate, Developer (hydroquinone, sodium sulfite, DI, acetone, Formaldehyde, pyridine, alcoholic gum mastic), Formic acid -Results: Spirochetes, bacteria-brown to black, background-pale yellow or tan
Steiner Procedure for Spirochetes, Campylobacter, and Legionella Orgs
-demo spirochetes, campylobacter pylori, or causative orgs of legionnellosis -Fix 10% NBF, avoid mercurial and chromium fix, paraffin 4-5um, QC-tissue containing spirochetes, C. Pylori, or Legionella orgs -Reagents: Uranyl Nitrate (sensitize), Silver Nitrate, Reducing Soln (Gum Mastic, Hydroquinone, Absolute alcohol) -results: spirochetes, c. pyloris, L.pneumophila and other nonfilamentous bacteria-dark brown to black, background-light yellow
Alcian Blue, pH 1.0
-demo sulfated mucosubstances -Fix in 10% NBF or Bouin's, paraffin 4-5um, QC-a section of small intestine, appendix, or colon -Reagents: HCl, Alcian Blue, Nuclear Fast Red -Results: sulfated mucosubstances-pale blue, background-pink to red
Gomori's Methanamine Silver Method for Urates
-demo urates -Fix: absolute alcohol is required, paraffin 4-5um, QC-sxn containing urates -Reagents: Methanamine-silver nitrate soln (silver nitrate, methanamine, sodium borate), Sodium thio, Light Green -Results: urates-black, background-green
Auramine-Rhodamine Fluorescence Technique
-detect TB or othera acid fast orgs -Fix 10% NBF, paraffin 4-5um, QC-tissue containing acid-fast myobacteria -Reagents: Auramine-Rhodamine Soln (auramine O, rhodamine B, glycerol, phenol, DI), Acid Alcohol (diff), Potassium Permanganate (counter) -Results: Acid Fast orgs-reddish yellow fluorescence, background-black
Rhodanine Method for Copper
-detect copper in tissue, especially in Wilson's liver disease -Fix 10%NBF, paraffin 6-8um, QC-a sxn with copper -Reagents: Rhodanine Soln (rhodanine, absolute EtOH, DI), diluted Mayer's Hematoxylin, Sodium Borate -Results:Copper-bright red to red yellow, Nuclei-light blue
Prussian Blue Stain for Ferric Iron
-detect ferric iron in tissues (normally found in the bone marrow and spleen)-large amounts are see in hemochromatosis and hemosiderosis -Fix in alcohol or 10%NBF, paraffin 4-5um, QC-sxn containing ferric iron -Reagents: 2% Potassium Ferrocyanide, 2% HCl, NFR -Results: nuclei and hemofuchsin-bright red, hemosiderin-blue, background-pink
Turnbull's Blue Stain for Ferrous Iron
-detect ferrous iron in tissues -Fix in alcohol or 10% NBF, paraffin 4-5um, QC-a section containing ferrous iron must be used -Reagents: HCl, Potassium Ferricyanide, Acetic Acid, NFR -Results: Ferrous Iron-blue, background-pink to red
Fite Acid-Fast Stain for Leprosy Orgs
-detect myobacterium leprae -Fix 10%NBF, or anything but Carnoy's, paraffin 4-5um, QC-tissue containing leprosy orgs -Reagents: Xylene-peanut oil (depar), Acid-alcohol (diff), Zeihl-Neelsen Carbol-Fuchsin Soln (Phenol crystals, alcohol, basic fuchsin, di), Methylene blue (stain, GAA)-counter -Results: Acid-Fast bacteria-bright red, background-light blue
Kinyoun's Acid-Fast Stain
-detect presence of acid-fast myobacteria in tissue sxns -lipoid capsule of an acid fast cell takes up carbol-fuchsin and resist decolorization with dilute mineral acid -Fix 10%NBF, others with the exception of carnoy's may be used, paraffin 4-5um, QC-tissue with acid fast orgs -Reagents: Kinyoun's Carbol-Fuchsin Stain (basic fuchsin, phenol crystals, alcohol, water), Acid Alcohol (diff), Methylene blue soln (counter) -Results: acid fast bacteria-bright red, background light blue
Masson's Trichrome Stain
-differentiate between collagen and smooth muscle in tumors, and to id increase in collagenous tissue in diseases such as cirrhosis of the liver -3 dyes, which may or may not include a nuclear stain, are used -Fix: Bouin's preferred, but 10% NBF may be used, paraffin 4-5um, QC-every tissue has an internal control, so no others are needed; uterus, small intestine, appendix or fallopian tube are good if one is desired -Reagents: Bouin's solution (picric acid, formaldehyde, GAA), Weigert's Iron Hematoxylin, Biebrich Scarlet-Acid Fuchsin Solution (BS, AF, GAA), Phosphomolybdic-Phosphotungstic Acid Solution, Aniline Blue Solution (Analine Blue, GAA, DI), Acetic Acid Solution -bouin's-mordant, -Results: Nuclei-black, Cytoplasm, Keratin, muscle fibers-red, collagen and mucous-blue
Alcian Blue-PAS-Hematoxylin
-differentiate between neutral and acidic mucosubstances -acidic are stained by alcian blue, and neutral are stained by PAS -Fix in 10%NBF or Zenker's, paraffin 4-5um, kidney cut at 2-3um, QC-kidney or mucin control -Reagents: acetic acid, alcian blue pH 2.5, periodic acid, Reducing rinse (sodium metabisulfite, DI water), schiff rgt -Results: exclusively acid mucosubstances-blue, neutral polysaccharides-magenta, certain substances will be colored by both PAS and alcian blue-purple
Alcian Blue with Hyaluronidase
-differentiate epithelial and connective tissue mucins -staining will be reduced in everything but glycoproteins -Fix in 10% NBF, 2 paraffin sections at 4-5um, one without digestion and the other with digestion, QC-2 sections of umbilical cord and a section of small bowel, appendix, or colon may be used as a 2nd control to demo epithelial mucins -Reagents: Buffer soln pH 6.0 (Potassium phosphate mono, sodium phosphate dibasic), Hyaluronidase digestion soln (testicular hyaluronidase, buffer soln), Alcian Blue, NFR -Without digestion: acid mucopolysaccharides and sialomucins-deep blue -With digestion: mucosubstances containing hyaluronic acid and chondroitin sulfates A and C-marked loss of stain
Thioflavin T Fluorescent method
-good for amyloid -thioflavin T is a fluorescent dye that attaches to amyloid and requires no differentiation -Fix in 10% NBF, paraffin 6-10um, QC a section containing amyloid Reagents: Thioflavin T solution, Acetic Acid (diff), Mayer's Hematoxylin (quench nuclear fluorescence) -Resutls: amyloid fluoresces yellow to yellow green
Schmorl's Technique for Reducing Substances
-indicates reducing substances in tissue. melanin, argentaffin granules and formalin pigment will stain - Fix in 10%NBF, paraffin 4-5um, QC-a sxn containing melanin or argentaffin granules must be used -Reagents: Ferric chloride-potassium ferricyanide soln, mayer's mucicarmine soln, metanil yellow Results: reducing substances-blue green, Goblet cells and mucin-rose, background- yellow green
ABC-Immunoperoxidase
-localization of tissue Ag -3 Reagents: primary Ab, biotinylated secondary Ab, and ABC (enzyme peroxidase, avid biotin complex) -Fix 10% NBF, B-5, Zenker's, or Bouin's; paraffin 4-5um, on poly-L-lysine-coated or silanized slides, dry overnight -QC-sxn positive for Ag, a negative control substituting buffer or nonimmune serum for primary should also be run -Reagents: ABC kit, PBS, Primary Ab, AEC, Acetate buffer -A positive reaction will be bright red
Basic Peroxidase-Antiperoxidase Immunoperoxidase Procedure
-localization of tissue Ags -Uses 3 reagents: primary Ab (specific for Ag), secondary Ab (links primary Ab to PAP), and a PAP complex -Fix: B-5, Zenker's, and Bouin's do not require trypsinization, but some Ags are not well demoed after fix in these. Some formalin-fixed Ags are better w/o trypsin, but many Ags are masked by formalin fix and will require it. -Paraffin 4-5um, on poly-L-lysine-coated or silanized slides, dry overnight -QC-sxn positive for Ag, a negative control substituting buffer or nonimmune serum from the same animal species as a primary should also be run -Reagents: PBS Solution (potassium phos di, sodium phos, NaCl, DI), primary Abs, swine anti-rabbit linking serum, rabbit PAP, AEC, acetate buffer -Results: A positive reaction will be brick red
Immunoperoxidase Staining with the 3-step Indirect Method
-localize tissue Ag -secondary and tertiary Abs are conjugated with peroxidase, primary monoclonal Ab is specific for Ag (2* is rabbit anti-mouse immunoglobulin, 3* is goat anti-rabbit immunoglobulin)-peroxidase reacted with chromogen -Frozen sections are fixed in cold acetone, frozen sections at 2-3um, cytospin preps made and dried overnight -QC-prep positive for the Ag, a neg control using buffer or nonimmune serum instead of the primary Ab should be run also -Reagents: TRIS-sodium chloride wash buffer, BSA, monoclonal Abs, normal human serum, rabbit anti-mouse imm., goat anti-rabbit imm., AEC -A positive reaction will be brick red
Crystal Violet
-rapid screening method for amyloid (not as specific as congo red) -Fix in 10%NBF or alcohol, cut paraffin at 10-12um, QC-a section containing amyloid -Reagents: Crystal Violet soln (crystal violet, 95% alcohol, DI water, HCl), Modified Apathy's mounting medium (acacia, cane sugar, di water, NaCl, Thymol) Results: amyloid-purplish violet, other tissue elements-blue
Mayer's Mucicarmine
-stain epithelial mucin in tissue sections -Aluminum forms a chelation complex with carmine giving a net + charge allowing it to attach to acid groups of mucin -Fix in 10% NBF, paraffin 4-5um, QC: colon, small intestine, or appendix -Reagents: Mucicarmine working solution (carmine, aluminum hydroxide, ethyl alcohol, DI), Weigert's Iron Hematoxylin, Mentanil Yellow Soln -Results: mucin-deep rose to red, capsule of cryptococcus-deep rose to red, nuclei-black, other tissue elements-blue or yellow
Biotin-Avidin-Horseradish Peroxidase Procedure (Labeled Avidin-Biotin Technique)
-used for surface marking of lymphomas -3 reagents: primary Ab, biotinylated secondary Ab, and Avidin Conjugated with Horseradish Peroxidase-chromogen DAB is applied to develop and observable color -Frozen sxns fixed for 10 min in cold acetone, 2-3um freeze in isopentane -QC-a neg. control using buffer or nonimmune serum instead of primary Ab should be run with each panel -Reagents: PBS Soln, Monoclonal Ab to human cell surface Ag, Normal human serum, Biotin conjugated F goat anti-mouse Ig, Avidin-D conjugated with horseradish peroxidase, DAB, copper sulfate soln, methylene blue counter -Result: surface Ab will be demoed with brown rings
The specimen must not exeed____________in thickness
0.3 cm
The permissible exposure limit(PEL) for formaldehyde is currently
0.75 ppm
List two methods for freezing tissue
1) in a cryostat 2) in isopentane and liquid nitrogen
List three methods to determine the endpoint of decalcification
1) manual 2) chemical 3) radiographic
For this technique, the tissue shown should be sectioned at
1-2 nm
Microtome Angles: 1. Bevel (knife) 2. Knife Clearance 3. Knife Wedge
1. 27-32 degrees 2. 3-8 degrees 3. 15-23 degrees
group 3: glycoproteins (mucins, mucoid, mucoprotein, mucosubstances)
1. Neutral (ovimucoid-egg white), stomach mucin, Panera cell granules 2. Sialomucins Epithelial mucins
The small square to the left of the surgical number in the cassette ID area in the image is known as
A 2D barcode
What type of microscope is typically used to observe auramine-rhodamine stained sections
A florescence microscope
The type of microscope used to examine the section in the image was
A fluorescence microscope
The microscope used for the image most likely uses
A halogen lamp
The crystal violet stain for amyloid is
A polychromatic stain
The tissue type in the image is
A portion of kidney glomerulus
Schiff reagent is
A reduced solution of basic fuchsin
An organelle that causes the cytoplasm to show increased basophoilia is the
RER
Nissl substance is composed of
RER
The blue staining of the cytoplasm in the image is due to
RER
The cytoplasmic material stained rose in the image is
RER
The intense rose color shown by the cytoplasm of some cells in the image is most likely due to
RER
What cellular component is stained rose by the methyl green-pyronin technique
RNA
What is the function of: Hydroquinine
Reduction
The issue in the image can most likely be corrected by
Reembeddig the tissue
Series of sulfurous acid rinses following PAS function to:
Remove excess Schiff reagent, prevent false pigmentation
Hot chloroform does what?
Removes all lipids
The issue in the image can be corrected by
Retracting the block holder shaft
Once the issue in the image is seen the only way to obtain a complete section is to
Ribbon past the holes of tissue permits
This step will protect a solution that is used repeatedly from pH changes because of the introduction of water
Rinsing sections with acid before Alcian blue solution
This step will prevent nonspecific staining
Rinsing with acid after the Alcian blue solution
surface decalcification
Rough in to expose the tissue - Surface of the tissue is placed in 1% HCl for 15-60 minutes - This allows only several sections to be cut
Tissue gouged , chunk cut out - reason
Roughed in advancing too far
Explain lable S95-6142-2
S - surgical 95- year 6142 - case# 2 - Blocks #
Collagen is stained yellow in the image by
Safran
Control tissue for Alcian blue 2.5
Same as Mayer's mucicarmine: unautolyzed small intestine, appendix or colon
The tissue structure in the image is
a blood vessel
The chemical group in dyes the confers the property of color is called
a chromophore
Sections for immunofluorescence is typically done on
a cryostat
A section with chatter may be the result of
a dull blade
What causes parched earth artifacts on a section
a flotation bath that is too warm
Isopropanol
a good substitute for ethanol for dehydration during processing, but not for use in staining procedures
The combination of a dye with a mordant is called
a lake
The tissue shown in the image is a section of
a muscular artery
The large, rose stained structure shown in the image is
a neuron
Water has a pH equivalent to
a neutral solution
What type of solution should be used to calibrate the pH meter to allow for adjustments to the pH of NBF
a neutral solution
Mushy sections and sections with chatter are not the result of
a nick in the blade
What type of microscope is typically used to observe Congo Red stained sections
a polarizing microscope
When cutting sections from paraffin blocks, the most common cause of unsatisfactory sections is
a poor blade edge
Microscopic examination of an H&E slide shown nuclei with well defined chromatin patterns, crisp nuclear membranes, and very pale pink staining of the cytoplasm and erythrocytes. These results indicate
a probable pH problem with the eosin
The property on which the acid fast stain depends is its
ability to resist decolorization with dilute acids
What's the fixative for: urate crystal
absolute alcohol
Clark solution
absolute alcohol glacial acetic acid -lysis RBC mix just before use. Good for bloody cytology smears
A good substitute for 1 of the chemicals in the primary staining solution used to stain the purple fibers in the image is
acetaldehyde
Neither b-5 nor orth contain
acetic acid
neuclear staining is made more selective by adding what to the hematoxylin solution
acetic acid
Carnoy's solution
acetic acid, glacial -lysis RBC chloroform absolute ethyl alcohol will preserve nucleic acids, lysis RBC, dissolve lipids and is not recommended for subsequent AFB staining tissue fixed in this must be washed with 95% -100% alcohol for the first step of processing
When a lens for a light microscope has been corrected for 2 colors, it is said to be
achromatic
Sections for special stains have been accidentally stained with hematoxylin. to remove the hematoxylin, place sections in
acid alcohol
Romanowsky type stains are combinations of
acid and basic dyes
What pigment can be removed with alcoholic picric acid
acid hematin
If placed in a solution with a pH below the IEP, cytoplasmic proteins will be
acidophilic
noncoagulant fixatives
act by creating a gel that makes penetration by the subsequent solutions difficult. Includes formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide and potassium dichromate.
agitation
adding to each step of the processing solutions aids in reagents flow thru the tissues leading to decreased time necessary for good processing results.
proteins
additive fixatives can alter they're 3D shape by changing electrical charges at the site of attachment. Nonadditive, coagulant fixatives cause these to become insoluble by altering tertiary structure
Gendre Solution
alcohol, 95% saturated with picric acid -hydrolyze nuclei formaldehyde, 37-40% glacial acetic acid- lysis RBC
See Schedule: Formalin, 10% 2 hrs (no heat, vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) Alcoholic formalin 1 hr ( no heat, no vacuum) 95 % alcohol 1 hr (only vacuum) 95 % Alcohol 45 min (no heat, no vacuum) Absolute Alcohol 45 min (only vacuum) Absolute Alcohol 1 hr (no heat, no vacuum) Xylene 1 hr no heat, no vacuum) Xylene 1 hr (only vacuum) Paraffin 30 min (no vacuum) Paraffin 1 hr (no vacuum) Paraffin 1.5 hr (vacuum) If the above schedule began at 5 pm, and the power cut out at 11:45 pm, and remained off for 2 hrs, microtomy on some tissue might show
chatter at the edges of the section
Cutting a paraffin black too quickly can result in
chatter on the section
The fixative used by a lab is being changed from formalin to glyoxal. it will likely be necessary to
check for the correct staining of H pylori
Very weak staining is notes on a PAS stained control section of liver. one problem-solving action is to
check the Schiff reagent with formaldehyde
EDTA (ethylenediaminetetraacetic acid )
chelating agent employed in decalcification that does not damage tissues, and immunohistochemical and enzyme reactions can be performed after use, but it is very slow in action, and a 24-hour time frame is not sufficient. Optium pH7-7.4 good for oxidative enzyme stains
chelating agent
chemical compounds that react with metal ions to form a stable water soluble complex.
Both paraffin and water soluble wax blocks need what before sectioning
chilling
The Gridley stain uses
chromic acid and Schiff reagent
Both Chloroform and Cedarwood Oil are
clearing agents
When checking the pH of a staining solution the pH meter should be calibrated using a standard solution with a pH value
closest to that of the staining solution
Spherical or ovoid bacteria are classified as
cocci
Masson's Trichrome shows
collagen
The orange stained tissue component in the image is
collagen
The tissue component stained blue in the image is
collagen
The tissue component stained red in the image is
collagen
Another technique that would look almost identical to the technique in the image is
colloidal iron
Of the following, the technique shown in the image is most likely Schmorl technique for reducing substances PAS Alcian blue pH 1.0 colloidal iron
colloidal iron
The stain shown in the image is most likely
colloidal iron
What staining technique was most likely used in the image
colloidal iron
Schiff's reagent is
coloress
Romanowsky Dyes
combinations of eosin and methylene blue
monobasic and dibasic sodium phosphates
commonly used to buffer formaldehyde solutions for routine use
In the section of umbilical cord seen in the image, the blue staining is due to
connective tissue mucin
A ph 7.0 buffer solution is
considered a neutral solution
a ph 4.0 buffer solution is
considered an acidic solution
Luxol fast blue stained sections show dark blue gray matter and lighter blue white matter. This can be most easily corrected by
continuing the differentiation step
The section shown in the image was most likely
coverslipped improperly by mashing on the coverslip
Sections stained with the Luxol fast blue-cresyl echt violet technique show bluish purple myelin and a diffuse rose-purple background. The most likely explanation is that the
cresyl echt violet was not acidified
The fungal organism seen in the image are most likely
cryptococcus neoformans
The stain demoed in the image is
crystal violet
Fluid parrafin converts to a solid by
crystalization
When processed on a short cycle, tissue must be
cut thin during grossing
Papanicolaou technique shows
cytology smears
1-2 seconds
cytology smears should be fixed in:
Autolysis
decomposition of tissue by enzymatic action begins as soon as blood supply is interrupted.
many of the zinc formalin fixed biopsy specimens are hard and brittle and show microscopic chatter. This will result if the specimens are
dehydrated with >70% alcohol
The problem in the image could have been prevented by
dehydrating the sections more completely