stain theory

Question 1

what is the purpose of staining

Answer 1

provide a contrast between tissue components by
1. attaching dyes to the tissue
2. reacting the tissue with organic reagents to produce coloured products
3. depositing metals on tissue components to produce visible insoluble black products

Question 2

dye colour theory

Answer 2

molecules absorb radiation in visible region
electrons move in response to gaining and losing energy
energy levels are unique to each molecule

Question 3

what are some component of dyes

Answer 3

organic compounds
most are coal tar or benzene derivatives
are able to attach to a substance by chemical bonding

Question 4

dyes can be classified according to their

Answer 4

-chromophores
-classficiations in reference books
-colour index numbers
-manufacturing process

Question 5

chromophores are

Answer 5

chemical group that results in colour
- the more numbers = increased colour intensity

Question 6

an uncoloured molecule +chromophore =

Answer 6

chromogen

Question 7

auxochromes

Answer 7

attach dye to tissue
- anionic (acid, net neg)
- cationic (basic, net pos)

Question 8

chromogen + auxochrome

Answer 8

stain

Question 9

classification of dyes by reaction with tissue

Answer 9

• cationic dyes
• anionic dyes
• mordant dyes
• solvent dyes
• polychromatic dyes
• neutral stain solutions
• amphoretic dyes
• leuco dyes
• metachromatic dyes

Question 10

cationic dyes

Answer 10

attach to anionic groups in tissue
stain is basic
substrate is basophillic
stain is acidophillic
often used to stain nuclei, basophil, mast granules, cartilage matrix, cytoplasmic anionic groups

Question 11

anionic dyes

Answer 11

attach to cationic groups in tissue
stain is acidic; substrate acidophilic, stain basophilic
used to stain cytoplasm and extracellular structures

Question 12

mordant dyes have a

Answer 12

weak auxochrome
- they stain tissues indirectly

Question 13

dye lakes

Answer 13

highly basic and act as cationic dyes

Question 14

cheif mordant dyes:

Answer 14

iron and alum

Question 15

solvent dyes are also known as…

Answer 15

lysochromes

Question 16

what is the use of solvent dyes

Answer 16

solvent dyes lacks an auxochrome and are hydrophobic
- when applied to tissue with hydrophobic domain it will get into other hydrophobic areas, helps stains fats, dye will be incorporated with the fat

Question 17

examples of solvent dyes

Answer 17

oil red-o
suban black

Question 18

solvent dyes are termed

Answer 18

selective or preferential solubility

Question 19

polychromatic dyes

Answer 19

spontaneously form other dyes in solution
- use a single solution

Question 20

what is polychromatic dyes often used for

Answer 20

rapid diagnosis of cryostat sections

Question 21

examples of a polychromatic dye

Answer 21

methylene blue
is oxidized into azure a and azure b (blue and purpe)

Question 22

neutral stain solutions

Answer 22

made from the interaction of anionic and cationic dyese

Question 23

example of a neutral stain

Answer 23

romanowsky dye in hem

Question 24

leuco dyes

Answer 24

colourless clear put into tissue and will change colour due to an occuring chemical reaction
- chromophores are easily reduces
-chemical reaction occurs with reactive tissue groups
- usually easily reversible
eg. periodic acid schiff

Question 25

metachromatic dyes

Answer 25

dye certain tissue components a colour different than the colour of the dye itself - can be basic or cationic -aqueous mounting media is recommended - sulfation can induce metachromasia

Question 26

what are the tissue components in a metachromatic dye calles

Answer 26

chromotrophs

Question 27

what is the basis of a metachromatic dye

Answer 27

stain is based on the tendency to polymerize - when two or more dye molecules come close together, get a new wavelength of light absorptivity

Question 28

interaction of dye with tissue involves

Answer 28

physical factors and chemical factors (electrovalent bonds, covalent bonds, hydrogen bonds, van der waals forces, hydrophobic interaction)

Question 29

physical factors

Answer 29

combine by absorption - dimension of surface area - density - permeability - size of dye molecule

Question 30

electrovalent bonds

Answer 30

the electrostatic attraction of oppositely charged ions known as ionic, electrostatic, salt linkages disrupted by acids, weak bases, water, alcohol, or high concentration of electrolytes dissociation = differenciation

Question 31

covalent bonds

Answer 31

formed when two atoms share electrons strongeat and requires lots of energy to disrupt bond typical of organic chemicals

Question 32

example of covalent bond stain

Answer 32

schiff's reagent verhoeff's stain

Question 33

hydrogen bonds

Answer 33

bond due to attraction between lone pair of electrons on O or N and partially exposed nucleus of H - weaker than covalent or ionic bonds - affect staining in non aqueous solvents

Question 34

van der waals

Answer 34

electrostatic attractions that exist between electrons of one atom and nucleus of another - found in all staining systems to various degrees - very short range forces - weakest

Question 35

example of van der waals staining

Answer 35

staining of elastin by aldehyde fuchsin

Question 36

hydrophobic interactions

Answer 36

affinity of non polar molecules to one another - stained preparations must be mounted in aqueous mounting media - the dye must be more soluble in the substrate and the solvent

Question 37

metallic impregnation

Answer 37

uses silver in alkaline solution readily able to precipitate

Question 38

what are the 3 types of reactions to occur in a metallic impregnation stain

Answer 38

argentaffin argyrophil metallic substitution

Question 39

argentaffin

Answer 39

reactive groups are capable to reducing silver salts

Question 40

argyrophil

Answer 40

tissue elements are not sufficiently reactive to reduce silver - outside reducer is required

Question 41

metallic substitution

Answer 41

ion exchange`

Question 42

progressive staining

Answer 42

in each stain until desired effect achieved - linear (X amount of minutes)

Question 43

regressive staining

Answer 43

tissue is overstained - takes a long time for tissue to stain and then it is gently removed

Question 44

differenciation

Answer 44

selective removal of excess dye from a section

Question 45

method of differenciation

Answer 45

- washing in water - using of excess mordant - oxidizing agents - other dyes : dye competition

Question 46

knowing the nature of the dye is important for

Answer 46

troubleshooting - what is the pH of dye solution - how has fixation affected the method