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Nuclear & Cytoplasmic Staining Flashcards

1
Q

What is the difference between Hematein and Hematin?

A

One is a stain (hematein) and one is an exogenous pigment (hematin).

Hematein is an oxidized derivative of hematoxylin used in staining. Blue and less soluble in alkaline (basic) solutions; red and soluble in alcohol (acidic) solutions.

Hematin is a brown/black iron containing pigment generated from hemoglobin oxidation.

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2
Q

What is the definition of a mordant and given an example?

A

Mordants are substances or metals that act as a link between dye and tissue.

Aluminum in hemateins (hematoxylins)

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3
Q

What are the ingredients and their purpose in Ehrlich hematoxylin?

A

Dye: Hematoxylin
Mordant: Potassium alum
Solvent: 95% EtOH
Oxidizer: Sunlight/oxygen
Acid: Glacial acetic acid
Stabilizer: Glycerol
Additional solvent: DI water

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4
Q

What are the ingredients and their purpose in Harris hematoxylin?

A

Dye: Hematoxylin
Mordant: Potassium alum
Solvent: DI water
Oxidizer: Mercuric oxide
Acid: Glacial acetic acid
Stabilizer: None
Additional solvent: 95% EtOH

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5
Q

What are the ingredients and their purpose in Modified Harris hematoxylin?

A

Dye: Hematoxylin
Mordant: Ammonium alum
Solvent: DI water
Oxidizer: Sodium iodate
Acid: None
Stabilizer: None
Additional solvent: None

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6
Q

What are the ingredients and their purpose in Gill hematoxylin?

A

Dye: Hematoxylin (I: 2g, II: 4g, III: 6g)
Mordant: Aluminum sulfate
Solvent: DI water
Oxidizer: Sodium iodate
Acid: Glacial acetic acid
Stabilizer: None
Additional solvent: Ethylene glycol

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7
Q

What are the ingredients and their purpose in Delafield hematoxylin?

A

Dye: Hematoxylin
Mordant: Ammonium alum
Solvent: DI water
Oxidizer: Sunlight/oxygen
Acid: None
Stabilizer: Glycerol
Additional solvent: 95% EtOH

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8
Q

What are the ingredients and their purpose in Mayer hematoxylin?

A

Dye: Hematoxylin
Mordant: Ammonium alum
Solvent: DI water
Oxidizer: Sodium iodate
Acid: Citric acid
Stabilizer: Chloral hydrate
Additional solvent: None

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9
Q

What are the ingredients and their purpose in Weigert hematoxylin?

A

Dye: Hematoxylin
Mordant: Ferric chloride
Solvent: DI water
Oxidizer: Ferric chloride
Acid: None
Stabilizer: None
Additional solvent: 95% EtOH

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10
Q

Define: Absorption

A

Tissue is penetrated by, or absorbs, a dye solution and becomes colored without any other change or chemical reaction occurring

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11
Q

Define: Adsorption

A

The accumulation of stain by the surface of a tissue component. Influenced by the affinity of oppositely charged ions for each other (electrostatic attraction or bonding)

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12
Q

Define: Mordant

A

A reagent used to link the stain, or dye molecules, to the tissue. Many are metals, especially those linking hematein to tissue

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13
Q

Define: Basophilia

A

An acidic (anionic, negatively charged) substance that is easily stainable with basic (cationic, positively charged) dyes.

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14
Q

Define: Acidophilia

A

A basic (cationic, positively charged) substance that is easily stainable with acidic (anionic, negatively charged) dyes.

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15
Q

Define: Ripening

A

Oxidation, as in the ripening or oxidation of hematoxylin to hematein

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16
Q

Define: Auxochrome

A

The chemical group present in a dye that causes it to bind to certain tissue elements. Can develop a charge and thus bind to oppositely charged groups present in the tissue (e.g. amino, -NH2, and carboxyl (-COOH) groups

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17
Q

Define: Chromophore

A

The chemical grouping that bestows the property of color on a compound

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18
Q

Define: Polychrome

A

Single dye solution that stains tissue components different colors by a phenomenon other than metachromasia (e.g. a solution prepared from a dye that is not pure but contains several dyes. The variety of color in the tissue is due to the selective adsorption of the different dye components by various tissue elements)

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19
Q

Define: Cationic dye

A
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20
Q

Define: Anionic dye

A
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21
Q

Define: Lake

A

The combination of a mordant (e.g. aluminum) with a dye (e.g. hematein). The dye-lake can bind to tissue components and form an insoluble colored deposit

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22
Q

Describe chromatin.

A

The stainable substance in the nucleus. Both consist of chromosomes of DNA and attached proteins.

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23
Q

Differentiate between heterochromatin and euchromatin.

A

○ Heterochromatin: contains condensed regions of chromosomes
○ Euchromatin: the extended portion of chromosomes (genetically active)

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24
Q

Which type of chromatin is stained by hemalum solutions

A

Heterochromatin
○ under the electron microscope, it is seen as collections of rounded or irregular shaped dense granules
○ under the compound microscope, it is seen as intensely basophilic nuclear material

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25
Describe how hemalum solutions is thought to bind to nuclear solutions.
26
Describe how cell activity affects the appearance of hematoxylin-eosin stained sections.
27
Differentiate between regressive and progressive staining.
28
State which of the hematoxylin solutions can be used for regressive staining.
29
State which of the hematoxylin solutions can be used for progressive staining.
30
Describe the theory involved with "physical" staining and give examples
Dye is absorbed (soaked up) by, and dissolved in, the cell. ○ Fat stain is physically absorbed by the lipids
31
List 4 methods of differentiation and describe the mode of action.
1. Basic or cationic dyes are differentiated by weak acidic solutions, and acidic or cationic dyes are differentiated by weak alkaline solutions. Differentiating solutions prepared in alcohol rather than water provide better control 2. An excess of mordant will break down the tissue-mordant-dye complex. Because the amount of mordant in the differentiating solution is large compared with that bound to the tissue, the dye will dissolve from the tissue. The structures that have bound the most dye will be the last to completely lose color. Careful control of the differentiating process will leave the desired structures well stained and the background colorless 3. Oxidizers work by oxidizing (bleaching) the dye to a colorless substance where those substances containing the most dye will still remain colored if the oxidation process is stopped at the appropriate time 4. Solvents are a simple and effective way to remove excess dye from tissue components
32
Give an example of using basic and acidic dyes for differentiation.
○ Aluminum hematoxylin can be differentiated with a dilute solution of hydrochloric or acetic acid ○ Eosin can be removed from overstained sections with dilute solution of ammonium hydroxide
33
Give an example of using excess mordant for differentiation.
○ Sections stained with regressive iron hematoxylin methods are differentiated with excess mordant (VVG)
34
Give an example of using oxidizers for differentiation.
○ Potassium permanganate and chromium trioxide (chromic acid) are oxidizing differentiators
35
Give an example of using solvents for differentiation.
○ Routine H&E - alcoholic eosin staining is followed by >1 solutions of dilute alcohols to obtain the differential effect of eosin ○ Aqueous eosin solutions may be lightly rinsed with water to obtain the same effect
36
List 4 natural dyes and their sources.
37
State why iron hematoxylin is the preferred nuclear stain in some staining techniques.
38
List the types of metal salts used as mordants in hemalum solutions and how each affects the color of the staining solution
39
State how excess of wither acid or aluminum in hematoxylin solutions can affect nuclear staining.
40
Describe the effect of pH on routine H&E staining.
41
List 3 factors other than pH that affect staining.
42
Describe the effect of overexposure to acidic fixatives or other acidic solutions on staining.
43
State how fixatives such as formalin and Bouin solution affect possible sources of error in routine H&E staining, the cause(s), and the appropriate corrective action for each.
44
Identify 3 methods of staining the cytoplasm.
45
Identify the 2 types of nucleic acids the methods of staining them.
Deoxyribonucleic acid (DNA) & Ribonucleic acid (RNA) ○ Feulgen reaction: demonstration of DNA ○ Methyl green-pyronin Y: differentiates between DNA & RNA, primarily used to identify plasma cells or immunoblasts in tissue
46
Discuss acid hydrolysis and fixation for the Feulgen reaction.
○ Mild hydrolysis (addition of water): of DNA using hydrochloric acid, which rapidly removes the purine bases (adenine & guanine) but leaves the sugars and phosphates of DNA intact. Generates aldehyde groups that are demonstrated with Schiff reagent ○ The optimum hydrolysis time is fixative dependent: Bouin solution hydrolyzes the nuclei excessively during fixation; therefore, tissue fixed in Bouin solution is unsatisfactory for use with the Feulgen reaction
47
List 2 dyes other than hematoxylin that may be used for nuclear staining.
48
List 2 methods other than H&E that may be used for staining frozen sectioning for rapid diagnosis.
49
Describe Romanowsky stains.
Combinations of the basic dye (methylene blue) and the acid dye (eosin)
50
Identify 3 primary uses for the Romanowsky stains.
51
List 2 major classes of mounting media.
Resinous & Aqueous
52
Identify the advantages and disadvantages of aqueous mounting media.
53
Identify the indications for using aqueous mounting media.
When dehydrating and clearing will adversely affect the stain.
54
Describe methods of sealing coverslip edges.
Sealants like melted paraffin wax, glue, or fingernail polish are painted on to the edges of the coverslip and serve as both a moisture barrier and to help adhere the coverslip to the slide.
55
State why coverslip edges may need to be sealed.
To prevent the section from drying or to preserve it better for longer.
56
Identify the advantages of resinous mounting medium.
57
Identify the disadvantages of resinous mounting medium.
Will cause gradual fading of the blue component of the Romanowsky stains
58
Compare natural and synthetic resinous mounting media.
59
Identify the indications for the use of resinous mounting media.
60
Identify 2 common solvents for resinous mounting media.
Xylene & Toluene
61
Discuss the types and thickness of slides and coverslips.
62
State the approximate refractive index range for resinous mounting media and relate it to the tissue.
1.51-1.55 ○ much closer to the average RI of tissue (1.53-1.54) --> as the RI of mounting medium approaches that of the tissue, the tissue becomes for and more transparent
63
State the approximate refractive index range for aqueous mounting media and relate it to the tissue.
1.41-1.49 ○ differs greatly from that of tissue, making microscopic evaluation more difficult.
64
Identify 3 coverslipping/mounting problems.
1. Water bubbles noted in mounted sections (f the sections are not completely dehydrated before clearing in xylene) 2. All areas of section cannot be brought into focus (mounting medium on top of the coverslip) 3. Cornflaking or drying artifact seen on mounted sections (sections are allowed to air dry before mounting creating a drying artifact - may appear as small individual cells seen as glossy block nuclei or as a large area of granular brown stippling resembling pigment) 4. Mounted stained sections are not as crisp as usual when viewed microscopically 5. Retracted mounting medium: a warped coverslip is applied or when the mounting medium has been thinned too much by xylene, or most common, when a tiny contaminant does not allow the coverslip to sit flat
65
Give the appropriate corrective action for water bubbles noted in mounted sections.
○ Change all dehydrating and clearing solutions before staining any more sections ○ Remove the coverslip and mounting medium with xylene and return to fresh 100% alcohol (several changes) and then clear with fresh xylene and mount with synthetic resin
66
Give the appropriate corrective action for when areas of section cannot be brought into focus.
○ Ensure mounting medium in not present on the top of the coverslip ○ Remove coverslip and remount section with a clean coverslip
67
Give the appropriate corrective action for Cornflaking.
○ Ensure slides do not dry out before mounting ○ Soak the mounted slide in xylene and recoverslip ○If severe, remove the coverslip and mounting medium with xylene; taking back to water to rehydrate, and then re-dehydrating and clearing
68
Give the appropriate corrective action for mounted stained sections not as crisp.
○ Ensure the mounting medium is not too thick, thus holding the coverslip too far above the section
69
Give the appropriate corrective action for retracted mounting medium.
○ Identify and carefully remove any contaminants keeping the coverslip from lying flat ○ Optimize the automated coverslipping process and perform regular maintenance ○ Remove and replace coverslip ○ Keep the container of mounting medium tightly capped when not in use ○ Discard the mounting medium when it becomes too thick
70
What is the source of the problem shown in this H&E?
Spotty or irregular staining is seen when water is left in the tissue during processing incomplete drying Not leaving slide in xylene long enough Paraffin saturated or contaminated depariffinization solutions
71
The problem seen in this H&E can be prevented by:
• ensuring section is properly dried before starting depar • ensuring enough time allowed in xylene • ensuring xylene is not contaminated - change solution regularly • decolorize and restain if problem is recognized after staining
72

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