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Histology Spring 2020 > Chapter 6: Nuclear and Cytoplasmic Staining > Flashcards

Chapter 6: Nuclear and Cytoplasmic Staining Flashcards

1
Q

Nuclear staining done with basic dyes

A

Cationic dye that forms salt linkages with DNA or RNA

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2
Q

Nuclear staining done with dyes and mordants

A

Dye is combined with, or followed by, a metal mordant, which helps link the stain to the tissue

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3
Q

What is Isoelectric point (IEP) with respect to pH

A

IEP is the point at which positive and negative charges are equal, this is independent of pH
This is important because most stains depend on ionic, covalent, or hydrogen bonds to bind the dye to the tissue
Thus the pH of a stain can be adjusted to moderate its effect
Proteins are cationic below IEP pH6, anionic above IEP pH6
so eosin (anionic -) is kept at pH5 so proteins remain cationic (+) when staining cytoplasm

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4
Q

Chromophore

A

Organic structures that confer color to a dye

Are easily reduced due to an affinity for Hydrogen(+)

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5
Q

Auxochrome

A

An ionizing group that enables the dye to link to tissue (can be on the dye or the mordant)

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6
Q

Mordant

A

A metal that helps bind the dye to a tissue

ex: aluminum, tungsten, iron, chromium, lithium

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7
Q

Lake

A

Dye + mordant = insoluble dye lake

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8
Q

Cationic Dye

A

Charge on the dye is positive
auxochrome is amino group NH2
Typically chloride salts

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9
Q

Anionic Dye

A

Charge on the dye is negative
auxochrome is sulfonic, carboxyl COOH, or hydroxyl OH
typically sodium salts

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10
Q

Amphoteric Dye

A

Cationic(+) if pH is below IEP, Anionic(-) if pH is above IEP
acidic environment causes the dye to be basic, basic environment causes the dye t be acidic
IEP varies by dye used, the iEP value is a pH
hematein

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11
Q

Acid Dyes

A

Charge on the dye is negative (-)
Auxochromes are sulfonic, carboxyl (-COOH), and Hydroxyl (-OH) groups
Eosin, acid fuchsin, picric acid, light green, Orange G

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12
Q

Basic Dyes

A

Charge on the dye is positive (+)
Auxochrome is the Amino group (-NH2)
Alcian blue, crystal violet, safranin

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13
Q

Progressive Staining

A

Dye up to the desired intensity, then stop the reaction

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14
Q

Regressive Staining

A

tissue is intentionally overstained, then decolorized with a differentiator to achieve desired stain intensity on the element of interest
Commonly used for mordant dyes

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15
Q

Feulgen Reaction Purpose

A

Demonstrate DNA

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16
Q

Feulgen Reaction Principle

A

HCl hydrolyses DNA removing the purine (A and G, pure as gold) bases, while leaving behind sugars and phosphates
The resulting aldehyde (-CHO) can be demonstrated with Schiff reagent

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17
Q

Feulgen Reaction Fixative

A

Avoid strong acid fixatives like picric acid (Bouin’s) because they will overhydrolyze the DNA
Any fixative without a strong acid is appropriate

18
Q

Feulgen Reaction Basic Procedure

A
  1. Deparaffinize (xylene) and hydrate
  2. Rinse in 1N HCl (displace remaining xylene/water)
  3. Heat in 1N HCl for 8-12 minutes (Hydrolysis)
  4. Rinse in cold 1N HCl then in distilled water (stops hydrolysis)
  5. Stain in Schiff reagent for 1 hour (dye binds to aldehydes)
  6. Wash briefly
  7. Rinse in 3 changes off sulfurous acid (reducing agent deactivates any residual Schiff)
  8. Wash in running tap water for 5 minutes
  9. Counterstain with 1% aqueous light green
  10. Dehydrate, clear (xylene), coverslip
19
Q

Feulgen Reaction Results

A

DNA (nuclei) is reddish purple

Counterstained cytoplasm is light green

20
Q

Feulgen Reaction Technical Notes

A

The Feulgen reaction must be carefully monitored because it has 2 phases, if the reaction runs too long you will get a false negative result

  1. removal of purine bases and formation of aldehyde (-CHO) groups
  2. removal of histones and apurinic acids

Duration of Feulgen reaction is dependent on fixative used

Counterstain must be applied lightly to avoid masking the Feulgen reaction

21
Q

Methyl-Green Pyronin-Y Purpose

A

Differentiate between DNA and RNA

Identify plasma cells or immunoblasts in tissues (smears, cores, aspirates)

22
Q

MGP Principle

A

DNA and RNA have different degrees of polymerization which results in differential staining
DNA stains with Methyl Green
RNA stains with Pyronin (Rose)
Methyl Green outcompetes pyronin for DNA binding sites

23
Q

MGP Preferred Fixative

A

10% Neutral buffered formalin
B-5, B+, Zenker, and Helly are also acceptable
Carnoy is the best

24
Q

MGP Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain with methyl green pyronin-y solution for 5 minutes
  3. Rinse with distilled water
  4. Blot dry with filter paper
  5. Dip in 2 changes of acetone
  6. Dip in 1:1 acetone:xylene
  7. Dip in 2 changes Xylene
  8. Incubate in Xylene 5 minutes
  9. Coverslip
25
Q

MGP Results

A 
DNA:  green to blue-green
Nuclei: Green to blue-green
Goblet cells: mint green
RNA: red to rose
Background: pale pink or colorless
Immunoblast and plasma cell cytoplasm: Intense RED
26
Q

Polychromatic stain

A

A compound dye or dye mixture that contains components of different colors
typically used for bone marrow specimens

27
Q

Romanowsky Stain

A

Polychrome stain category commonly used in pathology of blood and bone marrow samples

28
Q

May-Grunwald/Giemsa Stain Purpose

A

Differentiate cells in blood generating tissues, esp white blood cells, and demonstrate some microorganisms

29
Q

Giemsa Principle

A

“Neutral” dyes combine basic(+) methylene blue with acidic(-) eosin resulting in a wide range of colors for staining blood smears
Often used to demonstrate white blood cells

30
Q

Giemsa Preferred Fixative

A

Zenker or B-5 (mercuric)

10% NBF is acceptable

31
Q

Giemsa Basic Procedure

A
  1. Deparaffinize and hydrate
  2. 5-10 minutes in Lugol Iodine to remove mercury from B-5/Zenker fixative, wash in tap water, treat with sodium thiosulfate, wash in running water, wash in distilled water
  3. 3 minutes 100% Methanol 2 times
  4. Stain in working Jenner solution for 5-6 minutes
  5. Transfer to working Giemsa for 45 minutes
  6. Rinse quickly
  7. Differentiate in 1% acetic water
  8. Rinse in distilled water
  9. Dehydrate, clear, coverslip
32
Q

Giemsa Results

A

Nuclei: blue
Bacteria: blue
Leukocyte cytoplasm: pink, gray, blue depending on cell type and development

33
Q

Giemsa Technical Notes

A

Working solutions are not stable, make fresh and discard for each use

pH of stain solutions are critical, optimize for the fixative being used
optimal pH of 6.4-6.9

34
Q

Difference and preference between Synthetic and Natural Mounting Resins

A

Naturals are acidic, turn yellow, cause stain fading over time, and dry very slowly
Synthetics have the ideal refractive index, are neutral, dry quickly and don’t yellow with age

35
Q

What is Aqueous mounting media and when is it used?

A

Used when dehydrating and clearing will adversely effect the stain (like Oil red O for lipids)
Has a lower refractive index so tissue doesn’t appear as transparent

36
Q

Refractive index and relation to transparency

A

Refractive index measures the bending of light as it passes from one medium to another

You want the refractive index of your mounting media as close as possible to that of your tissue to maximize transparency and clarity

37
Q

Refractive index of tissue

A

1.53-1.54

38
Q

Refractive index of mounting medias

A

Synthetic Resin: 1.51-1.55

Aqueous: 1.41-1.43

39
Q

MGP Technical Notes

A

Ethyl green is better because you don’t have to purify it with chloroform

Carnoy fixed tissue yields the best results

Acids/some fixatives can depolymerize DNA preventing green stain

Pyronin isn’t specific to RNA, rather it is out competed for DNA by methyl green

Chloroform hazardous waste disposal

40
Q

5 Factors that affects dye binding

A
  1. pH affects charges on dyes and target molecules
  2. increased temperature tends to increase rate of staining
  3. Higher concentration of dye molecules tends to increase dye binding
  4. salts other than the dye can increase or decrease stain intensity of certain tissue components
  5. Fixative components can effect the ability of dyes to bind

Histology Spring 2020 (14 decks)

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