Histotechnology HTL Exam - Comprehensive Review

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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


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