Chapter 6: Nuclear and Cytoplasmic Staining Flashcards
What type of tissue is Harris hematoxylin used to stain?
Nuclei.
How do you “ripen” hematoxylin?
You expose it to oxygen.
What is the active staining molecule in ripened hematoxylin called?
Hematein.
What is the most important step in regressive hematoxylin staining?
The differentiation in acid alcohol because regressive hematoxylin staining is overstaining then differentiating.
Hematein is formed in Mayer hematoxylin solution by the addition of:
Sodium Iodate in order to “ripen” (oxidate) the hematoxylin.
How are mordants used in stains?
Oxidize the staining solution.
During H&E staining, if ammonia is incompletely removed by washing, the result may be:
Poor eosin staining.
The combination of a dye and a mordant is called a:
Lake.
Mercuric oxide was used in the original formula for Harris hematoxylin to:
Form hematein, but it was extremely toxic so it was replaced.
Which dye can demonstrate DNA?
The Feulgen reaction as well as methyl green.
T/F: Weigert is considered an Iron hematoxylin.
True.
What kind of hematoxylin is not readily decolorized with acidic solutions?
Iron hematoxylins, such as Weigert.
Why are natural resins hardly used for mounting sections?
Natural resins such as Canada balsam and gum dammar are slightly acidic and cause tissue fading after several years.
What kind of stain requires the use of an aqueous mounting media?
Oil red O so it doesn’t disrupt the lipid laden macrophages.
What type of metal salt serves as the mordant in Weigert hematoxylin?
Iron
Why is acetic acid added to Harris hematoxylin?
To make nuclear staining more specific.
What is a dye that can be substituted for hematoxylin in routine staining?
Celestine blue can be substituted for hematoxylin with identical results.
What is a good example of a polychrome stain?
Giemsa.
What is a polychrome stain?
A polychrome stain is a compound dye or dye mixture containing components of different colors.
What cellular structure is stained rose by the methyl green-pyronin (MGP) technique?
RNA.
The chemical group in dyes that confers the property of color is called a:
Chromophores.
What is an auxochrome?
Ionizing groups that confer to the tissue.
What is formed when hematoxylin is subjected to the action of sodium iodate?
Hematein.
What is heterochromatin stained by?
Heterochromatin is the substance in the nucleus that is stained by hematoxylin.
What stain is a good example of dye absorption?
The oil red O because all of the dye is soaked up by the lipids.
If placed in a solution with a pH below the IEP, cytoplasmic proteins will be:
At a pH of below 6.0, the cytoplasmic proteins will develop a dominance of positive charges and will attract acid dyes.
How do you differentiate the hematoxylin in H&E stains?
A weak acid (acid alcohol).
A tissue component that takes up cationic (positively charged) dye is said to be:
Basophilic.
What dye category does eosin fall into?
An anionic (acidic) dye.
For best cytoplasmic staining, the pH of eosin should be in between:
4.6 and 5.0.
What is the function of ethylene glycol in hematoxylin solutions?
It prevents a surface sheen.
What hematoxylin stain will stain the mucin in goblet cells?
Gill, because it does not have an excess of aluminum ions to out compete the hematoxylin.
What cellular structures does the Feulgen reaction demonstrate?
DNA only.
The Feulgen reaction is unsatisfactory on tissue fixed in:
Bouin solution, because it hydrolyzes the nuclei excessively.
Romanowsky type stains are a combination of:
Acid and base dyes.
Nuclear staining is made more selective by adding what to the hematoxylin?
Acetic acid.
Hematoxylin is converted to a dye lake in the Mayer formula by adding what to the solution?
Either ammonium or potassium aluminum sulfate.
A large quantity of Delafield hematoxylin stock solution must be maintained because:
Delafield hematoxylin is oxidized by air and light so a large quantity has to be maintained because of how slow the oxidation process is.
To link hematoxylin to tissue DNA, what must be added?
A mordant.
When does hematoxylin officially become a dye?
Is oxidized.
Romanowsky type stains are preferred for the demonstration of:
Leukocytes.
For the most transparency and clarity when viewing well stained microscopic section, the refractive index of the mounting medium should be:
Near that of the tissue so the tissue will become more transparent.
Synthetic resins are preferred over natural resins because:
Synthetic resins harden more quickly, don’t cause fading of the tissues and do not yellow over time.
What is the approximate refractive index of aqueous mounting media?
Aqueous mounting media has a refractive index that is significantly below the tissue.
How can a decrease in section transparency be caused?
The transparency of the section decreases if the mounting media becomes too thick.
Fixation with NBF will cause the cytoplasm to:
Take up more hematoxylin because the formalin binds with the -NH2 groups which leaves less of these groups to bond with eosin.
If the pH is between 4.6 and 5.0, what chemical groups will the eosin bind to?
-NH3.
What can be added to aqueous mounting medium to prevent bleeding of aniline dyes into the surrounding medium?
Cane sugar.
A Giemsa is most satisfactory if the pH is between:
6.4 and 6.9.
What hematoxylin solution must be used within a few days after preparation?
Weigert.
What organelle causes the cytoplasm to show increased basophilia?
The rough endoplasmic reticulum because of the large amount of ribosomes in it.
T/F: Basic dyes have a negative charge.
False, they’re cationic or positively charged dyes.
T/F: Oxidizers are sometimes used for differentiation.
True.
T/F: Eosin is differentiated by the dehydrating alcohols.
True, especially the lower ones like 70%.
T/F: An increase or decrease in the pH of the staining solutions can alter staining by changing tissue/dye charges.
True.
T/F: Staining can be influenced by the fixative used.
True.
T/F: Gill hematoxylin is an iron hematoxylin.
False, Gill is an aluminum hematoxylin.
T/F: Ferric chloride is both a mordant and an oxidizer.
True.
T/F: An increase in temperature usually increases the rate of staining.
True.
T/F: Toluene Blue can stain frozen sections.
True.
T/F: Resinous mounting media can have an index of refraction much lower than that of the tissue.
False, the refraction index must be very close to that of the tissue.
T/F: Resinous mounting media are usually dissolved in toluene, xylene or a xylene substitute.
True.
T/F: “The more the better” is a good rule of thumb for using mounting medium.
False, too much mounting medium can blur the tissue.
T/F: The nucleolus of plasma cells is stained green with the methyl green-pyronin technique.
False, the RNA is stained by this technique.
T/F: Bouin solution is a good choice for tissue to be stained with the Feulgen technique.
False, it would excessively hydrolyze the nuclei.
Microscopic evaluation of an H&E stained section shows nuclei with well defined chromatin patterns, crisp nuclear membranes and very pale pink eosin staining of RBCs and cytoplasms. What happened?
Incomplete removal of the bluing will cause inadequate eosin staining, there is a pH issue with the eosin.
An H&E stained section shows reddish brown stained nuclei, pink cytoplasm and bright rose-red RBCs. This would indicate:
Overripened hematoxylin will cause this.
An H&E stained section shows very uneven staining, with some areas of the slide well stained with other areas unstained. What happened?
The paraffin was not completely removed during staining.
While staining a rack of sections, it is noted that the water following the rehydrating alcohols is very cloudy. What’s wrong?
The xylene has been carried over into the water, the alcohols need to be changed.
H&E stained sections reveal brown pigmentlike stippling and rare black nuclei. What happened?
The mounting medium has dried before coverslipping.
H&E sections of liver show very dark nuclei and some blue staining of the cytoplasm. What happened?
There was inadequate hematoxylin differentiation.
H&E stained sections show that RBCs, collagen and muscle are all stained the same color. What happened?
Poor differentiation of eosin, there should be three different shades of pink.
A blue-black precipitate is seen on H&E stained sections. This could be prevented in the future by:
Filtering the hematoxylin to avoid these crystals that are formed during the ripening process.
Water bubbles are seen during H&E evaluation. This can be prevented in the future by:
Making sure that the dehydration process has been completed.
Microscopic evaluation of a kidney section is very difficult. Some areas are difficult to focus on while others are perfectly clear. This is most likely because:
Mounting medium is on top of the coverglass.
H&E stained sections show hazy blue nuclei, but recuts from tissue processed a week earlier show excellent nuclear staining. One possible cause of this problem is:
The use of too much heat during processing.
A Feulgen stain is requested on a section of Bouin fixed lymph node. How can you accomplish this?
Tissue fixed in another solution should be requested as the nuclei has already been hydrolyzed excessively by the Bouin solution.
Sections for specials have accidentally already been stained with hematoxylin. How can you remove the hematoxylin?
Acid alcohol.
The last dehydrating 100% alcohol in the stainer is very pink. This would indicate:
Since the eosin is usually differentiated by the lower alcohol concentrations, this would indicate that this absolute alcohol has been contaminated with water.
How often should the solutions be changed on the stainer?
Solutions should be rotated based on the number of slides that will be going through the stainer.
Giemsa staining is very poor on a bone marrow specimen. This problem can be corrected in the future by:
The pH of the staining solution needs to be changed, that’s probably why we make our Giemsa solution from scratch every time.
Microscopic review of an H&E shows an artefact known as “cornflaking”. What happened?
The slide has been dried prior to mounting.
What color are mycobacteria stained during the Fite procedure?
Red.
What stain will bind to acid mucosubstances, which can be then demonstrated during the Prussian blue reaction?
Colliodal iron.
Duplicate sections are stained with PAS, one with diastase and one without digestion. When the staining results are evaluated, what will the digested section demonstrate?
Sites of glycogen removal.
What is an example of a birefringent pigment?
A formalin pigment.
Acid mucosubstances and neutral mucosubstances can be differentiated by staining with what dual stain?
Alcian blue and PAS.
In the colliodal iron stain, the principal of the reaction is believed to be the formation of an ionic bond between the ferric ion with the carboxyl and sulfate groups of:
Acid mucosubstances.
In order to suppress background and nonspecific staining, a Congo red solution frequently contains:
Sodium chloride.
A lymph node is stained with a silver method for Retic and counterstained with nuclear fast red. After dehydration, there is a film over the entire slide that remains through clearing and coverslipping. What happened?
The slides were not adequately rinsed with water after counterstaining.
A section of muscular artery had been stained with the Verhoeff-van gieson procedure. Microscopic evaluation shows blue-black nuclei, black elastic fibers with orange collagen and muscle. What happened?
The picric acid was too dilute so the muscle and collagen weren’t properly differentiated.
Yellow-brown pigment, often found in cardiac muscle and liver cells in increasing amounts with age or debilitated states is known as:
Lipofucsin.
Hemosiderin, hemoglobin and bile pigment are all classified as:
Endogenous pigments.
The most appropriate hematoxylin solution for nuclear staining in a lengthy procedure that uses very acidic solutions is one that is mordanted with:
Iron, because it will not be removed by acids.
A modified phosphotungstic acid-hematoxylin procedure can be used to demonstrate:
Glial fibers.
What is an example of an argyrophil method?
The Grimelius stain.
In the Bodian technique, interference with primary staining may occur with prolonged treatment in:
Oxalic acid will ruin the silver proteinate reaction in the Bodian technique.
The staining intensity of eosin is increased in muscle and RBCs when tissues are fixed in:
Zenker because it does not bind to cationic groups.
The lab has used all of the supply of aluminum hemaxotylin solution. In order to prepare a new solution for immediate use, the solution must be:
Chemically ripened (oxidation).
When using the cresyl echt violet method, Nissl substance and nuclei can be preferentially stained by varying the degree of differentiation and the:
Solution pH.
Delafield hematoxylin is most commonly aged with:
Light.
What is an amphoteric substance?
An amphoteric substance can act either as an acid or as a base depending on the pH of the solution.
Microscopic sections stained with H&E show a lack of nuclear staining even though the hematoxylin is well ripened and all of the other solutions are fresh. What happened?
The prolonged storage of the tissue in NBF.
Microscopic review of an H&E stain shows that the eosin is very pale. What happened?
There was inadequate rinsing after neutralization.
What is the staining mechanism in which dye adheres to the surface of structures?
ADsorption.
When is Weigert hematoxylin considered unsatisfactory?
After three to four days.
In a routine staining series, slides placed in xylene for clearing prior to coveslipping have an opaque appearance. What happened?
There was incomplete dehydration.
Tissue sections were stained for the recommended time with H&E using Harris hematoxylin. A QC check shows pale nuclear staining. What happened?
The slides spent too long in the differentiating solution.
T/F: Dyes used for nuclear staining are basic.
True.
A stock solution of Harris hematoxylin is being prepared for use in the routine H&E stain. To make it an effective stain, what must first happen to the heamtoxylin?
It must be oxidized (primed).
What is an antigenic determinant present on a complete antigenic molecule?
Epitope.
What is a dye that absorbs light and then emits its own light at a longer wavelength?
Flurochromes.
What is a fungus that can be demonstrated with various stains for acid mucins?
Cryptococcus neoformans.
ATPase stains are done on a muscle biopsy at pH 9.4, 4.6 and 4.3. The slides for pH 4.6 shows differentiation of type I and type II fibers but NOT type IIA and IIB fibers. What happened?
The pH should have been modified on the 4.6 stain.
What is the pathologic condition characterized by abnormal deposits of iron in the liver?
Hemochromatosis.
What is an enzyme technique that will demonstrate denervated muscle fibers and motor end plates?
alpha-naphthyl acetate esterase.
What procedure will demonstrate H. Pylori without the use of a microwave oven?
The Diff-Quik.
A research project requires the demonstration of lysosomes on frozen sections of muscle tissue. What stain should be used?
Acid phosphatase.
In the PAS reaction, how is the development of the final product achieved?
Restoration of the quinoid structure.
Mucicarmine positive material is noted in the cytoplasm of poorly differentiated malignant cells. What kind of cancer would this indicate?
Adenocarcinoma because mucicarmine is specific for epithelium.
What is the rare enzyme technique that can be performed on paraffin sections?
The naphthol AS-D chloroacetate esterase.
How will the activity of succinic dehydrogenase be destroyed?
Fixation.
What are structures that speed up the rate of enzymatic reactions?
Cofactors.
What are enzymes that catalyze chemical reactions in biological systems?
Proteins.
What is a procedure that demonstrates some carcinoid tumors as well as the alpha pancreatic cells?
The Grimelius stain.
A patient is suspected of having McArdle disease which is a glycogen storage disease. The disease is BEST demonstrated with which stain?
The phosphorylase technique.
Experiments have shown that a particular lipid fraction known as the mycolic acid exists within the cell wall of what bacteria?
Mycobacterium tuberculosis.
Ab molecules can belong to one of 5 immunoglobulin classes. What is the Ab class most commonly used in IHC?
IgG.
What causes the red-brown nuclear staining sometimes seen with hematoxylin?
The solution has begun to breakdown.
How can the excess background staining be broken down with IHC stains?
Pretreating with nonimmune serum from the same animal species as the secondary Ab.
What fungi may appear as a mixture of budding yeast cells and pseudohypha elements in infected tissue?
Candida albicans.
What are mycotic diseases that go beyond superficial or cutaneous involvement to affect vital organs called?
Systemic.
In a suspected case of alzheimers, what stain would be ordered?
A Bielschowsky stain.
What method would be used for the demonstration of rickettsias?
Giemsa.
What does the succinic dehydrogenase technique stain?
Mitochrondria.
What would make a good control for Ab Cam 5.2?
A section from a carcinoma.
A rhabdomyosarcoma is suspected in a biopsy submitted to the lab. To aid in making a definitive diagnois, what stain would be ordered?
A PTAH stain would be ordered.
In lung tissue, coated asbestos fibers can be made more visible by staining with which stain?
The Prussian blue reaction.
What kind of cell is responsible for immunoglobulin production?
Plasma cells.
Melanosis coli is a condition characterized by abnormal deposits of pigment that will give a positive reaction to which stain?
Schmorl.
Astrocytes can be demonstrated by using the Ab that is specific for what IHC test?
GFAP.
`What would be the best stain to demonstrate loss of muscle striations caused by dystrophic change?
The Mallory PTAH stain.
What is the amorphous transparent gel- like material that forms the bulk of extracellular content in connective tissue?
The ground substance.
What are the small eosinophilic inclusions found in the cytoplasm of neurons in patients infected with the rabies virus?
Negri bodies.
What is a fungal disease characterized by narrow-based budding and carminophilia of yeast organisms?
Cryotpcoccus.
What is a maltese cross?
It is a configuration that is produced in tissue sections by the polarization of talcum powder, or starch in general.
What is the pigment occurring in Plasmodium parasites that is closely related to formalin?
Malarial.
In IHC reactions using horseradish peroxidase, how is the solution of hydrogen peroxide in methanol used?
To block endogenous peroxidase.
In ICH staining of NBF fixed tissue, does the heat-induced epitope retrieval enhance primary staining?
Yiss.
Following IHC staining, both the positive control and the specimen show weak staining. What happened?
The epitope retrieval was incorrectly done.
