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Histology2 > staining > Flashcards

staining Flashcards

1
Q

ways to make things stand out

A

dyes& stains

chemical reactions

metallic deposits

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2
Q

what is a dye

A

a dye is an organic compound which contains
chromaphoric and auxochromic groups attached to benzene rings

chromophoric group : responsible for color

auxochromic group : capable of binding to the reactive groups in the tissue

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3
Q

chromagen

A

he colored part of a dye molecule

aromatic compounds containing chromophores

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4
Q

Chromophore

A

the arrangement of atoms within the chromogen that is responsible for light absorption in the visible spectrum

the double bonds in this arrangements of atomic linkages alternate with single bonds to form “ conjugated systems “

the bonding electrons are able to move from one atom to another exchanging the position of linkages, this is referred to as resonance

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5
Q

fluorochrome

A

a chromophore which absorbs UV light and emits light of a longer wavelength

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6
Q

Auxochrome or Colligators

A

responsible of attachment of a chromogen to a substrate

a side chain attached to a chromaphore group

  • ionizable
  • reacts to form covalent bonds with a substrate
  • forms bonds with a mordant
  • many dyes have more than one Auxochrome

auxochrome gives the compound the property of electrolytic dissociation

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7
Q

Chromophore groups

A

dyes aren’t classified but their color they are classified by their chromophore group

dyes with the same chromophore group don’t necessarily have the same color

certain atomic groupings are associated with color

color is caused by selective absorption of certain wavelengths of light so that the light transmitted from a given substances lacks these wavelengths

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8
Q

Auxochrome groups

A

allows chromagens to bind to tissue groups either electrostatically or by covalent bonding

they give the dye the ability to dissociate in the solution. this salt forming property gives the dye the affinity to attach to tissues

auxochromes in basic dyes: NH3+

auxochromes in acid dyes : OH or SO3-

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9
Q

lysochromes

A

not dyes in the true sense bc they lack auxochromes and do not ionize

they stain by selective or preferential solubility
- will color a substance in which they are soluble

fat staining is preformed in this manner

also referred to as simple solubility or selective solubility

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10
Q

colour index number

A

`dyes with the same name can have very different contents
and the same dyes can have different names
to avoid confusion the is a worldwide agreement of naming dyes with a standard numerical classification

this system is called Colour Index Number & is for the identification of pure dye* structures

5 digit #
allows for standardization of staining methods

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11
Q

biological staining commission

A

body responsible for standardization of biological stains
certification of a stain by the commission indicates:
1. A sample of the dye has been submitted for testing
2 Part of the sample is kept on file
3. The sample proves true to type by Spectrophotometric tests
4. The dye content is up to specification and is correctly indicated on the label.
5. It has been tested, by experts, in the procedures named and has been found
satisfactory.
6. No other batch of dye can be sold under the same Certification Number

Dyes which have been certified by the Biological Staining Commission should have the following information on the label:
The name of the dye
Lot number
Dye content 
Colour index number
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12
Q

Dye content

A

recipes for dye solutions are based on the stain being 100% dye which si rarely the case

present dye/ new dye = gravimetric factor

gravimetric factor x amount called for

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13
Q

natural dyes

A

derived from plants, animals & insects

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14
Q

synthetic dyes

A

referred to as coal tar dyes
includes most of the dyes we use in histology
majority are derived from benzene today***
dyes are generally carcinogenic

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15
Q

vitals staining

A

when living tissue is stained
not as common
ex. reticulocyte staining in hematology

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16
Q

post-mortem staining

A

most staining is preformed on fixed tissue

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17
Q

direct staining

A

dye is directly applied to tissue to accomplish staining

dye has great auxochromes

place dye & substrate together

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18
Q

indirect staining

A

staining process which requires a mordant to achieve staining

substance that requires a mordant to stain isn’t considered a sye until mordant is added

a mordant is a metal with a valency of at least 2

  • ‘dye’ is weak; has poor or low auxochromes
  • add metal to form a dye - lake
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19
Q

trapping agent

A

Trapping agents are chemicals which inhibit the removal of dyes from tissue

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20
Q

Negative staining

A

refers to making an object visible by staining the background
rather than the object to be viewed

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21
Q

Leuco Compounds

A

Although chromophores differ from one another in a number of ways; they are all easily reducible, that is combine easily with hydrogen. The reduction process will break the chromophoric grouping and the dye will lose its color.

This colorless compound is called a leuco compound. The reduction is reversible under oxidizing conditions

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22
Q

hemosiderin “staining”

A

Hemosiderin forms the body’s iron stores. The iron is in the ferric state, and may be demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. The iron reacts with potassium ferrocyanide to form ferric ferrocyanide. This is an insoluble, blue compound known as
Prussian blue

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23
Q

metallic impregnation

A

Referred to as part of special stains but no dyes are actually involved instead,
metallic ions are deposited on selected fibres and cells to provide contrast.

often refer to these as silver stains. Some metallic compounds can be
reduced by tissues to the metallic state, producing an opaque, usually black
deposit

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24
Q

metallic impregnation using silver nitrate : one stage reaction

A

The tissue substrate is able to reduce silver compounds to a black deposit
directly.

called Argentaffin Reactions

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25
Q

metallic impregnation using silver nitrate: 2 stage reactions

A

tissue substrate is unable to reduce silver compounds to a black deposit
directly. The method requires the application of a second chemical known as
an extraneous reducer

Unreduced silver is deposited on the tissue fibres and reduced to a black
deposit in a second reaction. Such reactions are called Argyrophilic

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26
Q

orthochromic staining

A

Staining reactions that impart the same colour to the tissue as the stain itself

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27
Q

metachromatic staining

A

ome staining reactions result in the staining of certain tissue elements
a different colour than the original colour of the dye. This phenomenon is
known as Metachromasia

change in colour is due to polymerization of the dye molecules by the tissue
chromatropes

*****dyes used in these reactions are generally cationic and include Toluidine blue and Methylene Blue

28
Q

polychromatic stain

A

single dye solution stains tissue components different colors without metachromasia being involved. The dye solution is not pure and therefore different tissue components selectively bind with different dye components producing different colors

29
Q

anionic and cationic dye relationships

A

A dye with a net positive charge is called basic or preferably cationic.
A dye with a net negative charge is called acidic or preferably anionic

“Acid” or “basic” dye (anionic, cationic) does not refer to the pH of the dye solution. refers to the salt of a colour acid/base

In electrostatic staining the dye and substrate are of opposite net
charge. Anionic binds with Cationic.

30
Q

acid dye

A

coloured anions, with sulphonic or carboxylic acid auxochromes
The acid component is coloured and the base is colourless
Acid dyes usually stain basic components, such as cytoplasm
Acid Dyes - Anionic - Negative Charge

31
Q

basic dyes

A

These are coloured cations with NH2+ auxochromes
The basic component is coloured and the acid is colourless with
Basic dyes usually stain acidic components, such as nuclei
Basic Dyes – Cationic - Positive Charge

32
Q

neutral dyes

A

Both the acidic and basic components are coloured.
They are prepared by combining aqueous solutions of Acidic and Basic dyes in specific proportions.
The resulting precipitate will be a Neutral dye
 Neutral dyes are soluble only in Alcohol (Methyl)

The staining reaction for these dyes takes place at ~pH 7.2 - 7.4 (slightly
alkaline)

33
Q

sulphonic group

A

can convert basic dye to acid dye

can convert an insoluble dye to a dye that is soluble in water

34
Q

Amphoteric

A

has both neg and pos charged groups

charge will depend on the pH of the staining solution

35
Q

basic auxochromes

A

Usually amines NH3

36
Q

acid auxochromes

A

Derived from sulphonic or carboxylic acid or from phenolic hydroxides

37
Q

progressive staining

A

staining until the desired staining intensity is achieved, then stopping

38
Q

regressive staining

A

over staining a tissue then differentiating( decolorizing) until only the desired element & intensity of staining is achieved

39
Q

decolorization vs differentiation

A

decolorization is technically removing all color

differentiation is removing color selectively to achieve the desired degree of colorization

40
Q

differentiation of a mordant is accomplished in 1 of 3 ways

A
  1. cationic dyes are differentiated with weak acids (opposite cahrge)
  2. an excess of mordant will break the bond between the dye
  3. using an oxidizer
41
Q

other means of differentiation

A
  1. solvents may be used to remove color
  2. other dyes may be used. this may be referred to as displacement
  3. blood smears stained with wrights stain are differentiated using buffer at pH
    6.5, at this pH the buffer acts as a basic differentiator of eosin & an acidic
    differentiator of methylene blue
42
Q

what makes dye stick

A

Affinity

  • the tendency of stain to move from solution into tissue
  • forces influencing stain-tissue & stain-stain interactions
  • van der Waals are the weakest & Coulombic are the strongest
43
Q

physcial forces of affinity

A

capillary

osmosis

absorption

adsorption ( both physical & chemical)

44
Q

capillary action

A

movement of liquid within a material against gravity as a result of surface tension

45
Q

osmosis

A

movement of water through a membrane

46
Q

Absorption

A

passing of dye molecules from the dye to the solution in the substance being dyed

fat staining involves non ionic dyes & is purely absorption

47
Q

Adsorption

A

adhesion of molecules to a solid surface resulting in heavily high concentration of the molecules at the place of contact

chemical action & salt linkages can also play a part in the adhesion

48
Q

physical considerations

A

size of dye particle
- large particles diffuse more slowly

porosity
-more porous substrates are easier to stain

interfacial effect
- dye concentration id higher at the surface of the solution & at the interface of the solution & walls pores

49
Q

chemical affinity : Electrostatic attraction

A

also referred to as coulombic attraction

involves the net charge of dyes & tissue components in a given solution

acidic dyes are basophilic
basic dyes are acidophilic

proteins in the cell are amphoteric; they depend on the pH of the solution
- they have a net charge of 0 at their isoelectric point, if placed in a pH lower than that they become positively charged ( basic)
in a pH higher than that they become negatively charged ( acidic)

50
Q

all of the following refer to the transfer of an electron from one to the other to form a bond

A

electrostatic attraction

electrovalent attraction

salt linkage

coulombic attraction

ionic bond

51
Q

other chemical aspects of dye binding

A

hydrogen binding
hydrophobic binding
van der Waals forces
Covalent bonds

52
Q

Electrovalent attractions

A

anionic dyes ( eosin) stain cationic tissue components ( proteins at a pH below 6)

cationic dyes ( hematin) stain anionic tissue components such as nucleic acids

53
Q

Salt linkage

A

Coulombic or electrostatic attractions are salt linkages

the amount of non dye salt in the solution will effect the amount of staining which occurs

54
Q

Van der Waals forces

A

weak electrostatic forces ( short distance)

attractions between the electrons of one atom and the nucleus of another

ex. elastin or some mordant staining

55
Q

factors influencing dye binding

A

ionic strength

dye concentration - greater amounts of dye may be bound from higher concentrations but is limited by the number of dye binding sites

fixation of tissue- generally following fixation affinity for dye increases more than unfixed tissue bc binding groups are more accessible to the dye

temperature - increase will cause an increase in staining ( increased diffusion rates & protein swelling

pH of the solution - affects dissociation of dye

thickness of tissue- more tissue binds more dye

56
Q

dye staining

A

H& E
most “special stains”
microbiology
hematology

57
Q

other “ staining methods “

A

metallic impregnation

selective solubility

immunohistochemistry

enzymatic stains

58
Q

immunohistochemistry

A

the main reagent is an antibody- generally monoclonal

may pinpoint the source tissue of a malignancy

59
Q

amine precursor & uptake decarboxylation (APUD) cells

A

diagnosis of neuroendocrine tumours

these cells demonstrate Argyrophil and/or Argentaffin reactions
- however now IHC is used as it provides more specific information

60
Q

direct immunohistochemistry

A 
fluorescein isocyanate (FITC) is applied to a frozen section containing glomeruli & examined using fluorescent microscope
this enables the detection & localization of immunoglobulins, complement components & fibrin

older and less sensitive than indirect method

is used for renal & skin biopsies

61
Q

indirect immunohistochemistry

A

testing for autoantibodies is usually done in this manner

patients serum is added to a tissue sample containing known antigens to test the patient for the presence of antibodies to these antigens

patients antibody is the primary antibody & attaches to the antigen in the tissue, secondary antibody is the antihuman immunoglobulin which attaches and can be detected due to its fluorescent tag

indirect can also mean that 2 antibodies are used to detect an antigen in the patients tissue, this can be performed with a two step or three step method
generally are enzymatic methods & detection can be performed using a light microscope

62
Q

enzymatic staining

A

testing for biological catalysts

hematology & histology

muscle biopsy

lymphoma & leukemia studies

63
Q

enzymatic tests

A

muscle biopsy snap frozen in liquid nitrogen
- with or without isopentane

lymph node carefully sliced
- touch preparation utilization

64
Q

enzymatic staining

A

special handling of specimens

touch preparations & frozen sections

DONT FIX SPECIMEN!

enzyme catalyses reaction with substrate

colored end product used for detection

65
Q

enzymatic testing varibles

A

pH

Temperature- may destroy enzymes

concentration of substrate & the enzyme

inhibitors
- heat, agitation, exposure to acid fixatives, others

activators
- divalent cations: magnesium, manganese & calcium

66
Q

types of enzymatic reactions

A

Azo dye method
- substrate contains one or more napthol compounds. enzyme activity releases the napthol which joins with a diazonium salt. The diazonium salts form reaction products to produce an azo dye which marks the reaction site

Indoxyl methods
- indoxyl substrates release indoxyl radicals that are rapidly oxidized to insoluble indigo blue

Metal salt methods
- used when one of the products of enzyme activity on the substrate is an acid. The acid is captured by a metal ion in the incubation medium & precipitated. A series of subsequent reactions ultimately produce an insoluble colored salt at the reaction site

oxidation - reduction reactions
- hydrogen is transferred from a substrate to a tetrazolium salt. the salt is reduced to insoluble purple formazam pigment at the site of activity

Histology2 (14 decks)

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