staining

Question 1

ways to make things stand out

Answer 1

dyes& stains

chemical reactions

metallic deposits

Question 2

what is a dye

Answer 2

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

Question 3

chromagen

Answer 3

he colored part of a dye molecule

aromatic compounds containing chromophores

Question 4

Chromophore

Answer 4

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

Question 5

fluorochrome

Answer 5

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

Question 6

Auxochrome or Colligators

Answer 6

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

Question 7

Chromophore groups

Answer 7

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

Question 8

Auxochrome groups

Answer 8

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-

Question 9

lysochromes

Answer 9

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

Question 10

colour index number

Answer 10

`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

Question 11

biological staining commission

Answer 11

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

Question 12

Dye content

Answer 12

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

Question 13

natural dyes

Answer 13

derived from plants, animals & insects

Question 14

synthetic dyes

Answer 14

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

Question 15

vitals staining

Answer 15

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

Question 16

post-mortem staining

Answer 16

most staining is preformed on fixed tissue

Question 17

direct staining

Answer 17

dye is directly applied to tissue to accomplish staining

dye has great auxochromes

place dye & substrate together

Question 18

indirect staining

Answer 18

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

Question 19

trapping agent

Answer 19

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

Question 20

Negative staining

Answer 20

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

Question 21

Leuco Compounds

Answer 21

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

Question 22

hemosiderin “staining”

Answer 22

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

Question 23

metallic impregnation

Answer 23

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

Question 24

metallic impregnation using silver nitrate : one stage reaction

Answer 24

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

called Argentaffin Reactions

Question 25

metallic impregnation using silver nitrate: 2 stage reactions

Answer 25

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

Question 26

orthochromic staining

Answer 26

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

Question 27

metachromatic staining

Answer 27

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

Question 28

polychromatic stain

Answer 28

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

Question 29

anionic and cationic dye relationships

Answer 29

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.

Question 30

acid dye

Answer 30

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

Question 31

basic dyes

Answer 31

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

Question 32

neutral dyes

Answer 32

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)

Question 33

sulphonic group

Answer 33

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

Question 34

Amphoteric

Answer 34

has both neg and pos charged groups | charge will depend on the pH of the staining solution

Question 35

basic auxochromes

Answer 35

Usually amines NH3

Question 36

acid auxochromes

Answer 36

Derived from sulphonic or carboxylic acid or from phenolic hydroxides

Question 37

progressive staining

Answer 37

staining until the desired staining intensity is achieved, then stopping

Question 38

regressive staining

Answer 38

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

Question 39

decolorization vs differentiation

Answer 39

decolorization is technically removing all color | differentiation is removing color selectively to achieve the desired degree of colorization

Question 40

differentiation of a mordant is accomplished in 1 of 3 ways

Answer 40

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

Question 41

other means of differentiation

Answer 41

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

Question 42

what makes dye stick

Answer 42

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

Question 43

physcial forces of affinity

Answer 43

capillary osmosis absorption adsorption ( both physical & chemical)

Question 44

capillary action

Answer 44

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

Question 45

osmosis

Answer 45

movement of water through a membrane

Question 46

Absorption

Answer 46

passing of dye molecules from the dye to the solution in the substance being dyed fat staining involves non ionic dyes & is purely absorption

Question 47

Adsorption

Answer 47

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

Question 48

physical considerations

Answer 48

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

Question 49

chemical affinity : Electrostatic attraction

Answer 49

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)

Question 50

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

Answer 50

electrostatic attraction electrovalent attraction salt linkage coulombic attraction ionic bond

Question 51

other chemical aspects of dye binding

Answer 51

hydrogen binding hydrophobic binding van der Waals forces Covalent bonds

Question 52

Electrovalent attractions

Answer 52

anionic dyes ( eosin) stain cationic tissue components ( proteins at a pH below 6) cationic dyes ( hematin) stain anionic tissue components such as nucleic acids

Question 53

Salt linkage

Answer 53

Coulombic or electrostatic attractions are salt linkages the amount of non dye salt in the solution will effect the amount of staining which occurs

Question 54

Van der Waals forces

Answer 54

weak electrostatic forces ( short distance) attractions between the electrons of one atom and the nucleus of another ex. elastin or some mordant staining

Question 55

factors influencing dye binding

Answer 55

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

Question 56

dye staining

Answer 56

H& E most "special stains" microbiology hematology

Question 57

other " staining methods "

Answer 57

metallic impregnation selective solubility immunohistochemistry enzymatic stains

Question 58

immunohistochemistry

Answer 58

the main reagent is an antibody- generally monoclonal may pinpoint the source tissue of a malignancy

Question 59

amine precursor & uptake decarboxylation (APUD) cells

Answer 59

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

Question 60

direct immunohistochemistry

Answer 60

``` 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

Question 61

indirect immunohistochemistry

Answer 61

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

Question 62

enzymatic staining

Answer 62

testing for biological catalysts hematology & histology muscle biopsy lymphoma & leukemia studies

Question 63

enzymatic tests

Answer 63

muscle biopsy snap frozen in liquid nitrogen - with or without isopentane lymph node carefully sliced - touch preparation utilization

Question 64

enzymatic staining

Answer 64

special handling of specimens touch preparations & frozen sections DONT FIX SPECIMEN! enzyme catalyses reaction with substrate colored end product used for detection

Question 65

enzymatic testing varibles

Answer 65

pH Temperature- may destroy enzymes concentration of substrate & the enzyme inhibitors - heat, agitation, exposure to acid fixatives, others activators - divalent cations: magnesium, manganese & calcium

Question 66

types of enzymatic reactions

Answer 66

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