Staining

Question 1

Van der waals forces

Answer 1

creates a significant binding force between the dye and protein although individually weak.
Strongest in large molecules as in Elastic fibre stains

Question 2

hydrogen bonding

Answer 2

does not play a major role in stain-tissue affinity as water is strongly hydrogen bonded to itself and inhibits hydrogen bonding while favoring ionisation

Question 3

ionic bonding, types of dyes

Answer 3

most important form of bonding in staining. negatively charged dyes are called anionic, and known as acidic dyes. Components which stain by acidic dyes are referred to as being acidophilic.
Tissues which recognize basic dyes are referred to as basophilic.

Question 4

Example of anionic/acidic dye and what is stains

Answer 4

eosin, stains erythrocytes, collagen, cytoplasm of many cells

Question 5

covalent bonds

Answer 5

not veru important in staining. seen in some hsitochemical stains such as Perioidic acid schiff

Question 6

What is a chromophore

Answer 6

chemical group which makes any organic compound coloured

Question 7

auxochromes

Answer 7

ionizable groups added onto chromogens to convert them into proper dyes which the tissue can retain

Question 8

metachromasia and orthochromasia

Answer 8

metachromasia - dyes which colour different tissue components different colours to the original colour of the solution. This is because it has the capacity to absorb light of more than 1 wavelength. eg. methylene blue, safranin

orthochromasia - staining of the same colour as the dye solution

Question 9

What chemical groups are in acidic and basic dyes and what they dye .

Answer 9

acidic -amino grousp,,mainly proteins in the cytoplasm and connective tissue
basic - used as nuclear stains, carboxylate, phosphtae,sulfate groups.

Question 10

neutral dye

Answer 10

basic and acidic dyes, with both ions being coloured therefore stain nuclei and cytoplasm. Such as Romanowsky stain: Giemsa.

Question 11

What does the mordant do

Answer 11

it gives great stability to the stain, is not easily removed by water, alcohol or weak acids and resists decolourization by other reagents. Overall it mediates the dye tissue interaction

Question 12

what can be used for chemical oxidation of haematoxylin

Answer 12

sodium iodate, potassium permanganate or mercuric oxide

Question 13

mordent of aluminium haematolxyins

Answer 13

• aluminium potassium sulfate or aluminium ammonium sulfate

Question 14

examples of Al haematoxylins

Answer 14

Harris’, Mayer’s, Gill’s, Ehrlich’s

Question 15

examples of iron haematoxylins

Answer 15

Weigert’s, Heidenhain’s, Verhoeff’s

Question 16

Addition of glacial acetic acid to Harris HAematoxylin

Answer 16

gives more precise and selective staining of nuclei

Question 17

How different fixatives affect staining with haematoxylin and eosin

Answer 17

formalin fixed tissue take up less eosin. Tixed fixed in solutions containing potassium dichromate tend to take up less haematoxylin

Question 18

What is histochemistry

Answer 18

The use of special stains to identify the location of chemicals within the tissues - which cannot be seen with H&E

Question 19

PAS stain componetns

Answer 19

–> Perioidic acid 1% - oxidises hydroxyl groups attached to adjacent carbon atoms within the carbohydrate or their amino derivatives to form two aldehyde groups

–> Schiff’s reagent - solution of basic fuchsin (pararosaniline) reacts with aldehyde groups resulting in the rearrangement of its sulphonic group and restoration of the chromophoric group producing deep magenta.

-> Harri’s haematoxylin used as a counterstain

Question 20

Components of MAsson’s Trichrome stain

Answer 20

• Bouin’s fixative: acts as a mordant and sets tissue at an acidic pH
• Biebrich Scarlet red : acidic stain for all componetns apart from nucleic acids.
• Weigert’s Iron haematoxylin: nuclear stain
• phosphotungstic/phosphomolybdic acid: removes excess biebrich scarlet and decolourizes collagen
• Aniline Blue: stains collagen blue

Question 21

How Verhoeff- van Gieson stain Elastin fibres stain works

Answer 21

Section is first overstained with Verhoeff stain. Dilute ferric chloride solution is used to break the tissue-mordant-dye complex. Elastic tissues have the strongest affinity for the insoluble compelx and retain it the longest - giving them a black colour. Van Gieson stain used as a counterstain and colours collagen bright red and other elements yellow

Question 22

Componetns of congo red and how they work

Answer 22

Pretretment with NaOH to release native internal hydrogen bonds.
NaCl to supress flase negative staining of collagen and elastin. important to carry out control.

Question 23

Other stains for amyloid

Answer 23

-crystal voilet polychromatic stain stains it purple
-toluidine blue dyes it blue
- sirius red stain stains it pink, green birefringence
- thioflavine reaction

Question 24

Dealing with sample with prion disease

Answer 24

fixed tissue and slides have to be inactivated by treatment in 96% formic acid for at least 1 hour.

Question 25

When is visualization of micro organisms in tissue sections useful

Answer 25

• multiple organisms are cultured
• none are recovered
• non cultivable agents
• a more timely diagnosis for diseases caused by organisms that are detected late because they grow slowly such as mycobacteria.

Question 26

Stains used for acid fast bacteria

Answer 26

Ziehl Neelson, Wade-Fite, Auramine/Rhodamine

Question 27

Stains for Spirochetes

Answer 27

Warthin and Starry, Toluidine Blue, Giemsa

Question 28

Stains for fungi

Answer 28

Grocott-Gomori, PAS

Question 29

What does acid fast mean

Answer 29

once the dye used to stain the bacilli is absorbed it is not removed when incubated in an acid rinse. E.g. Mycobacterium tuberculosis and Mycobacterium leprae

Question 30

how are fungi detected by staining

Answer 30

through oxidation of their rich polysaccharide walls into dialdehydes.

Question 31

How does grocott’s methenamine silver stain work

Answer 31

chromic acid oxidises hydroxyl groups of cell wall polysaccharides into aldehyde groups. Aldehyde oxidation products reduce the silver nitrate to metallic silver therefore rendering them visible.

Question 32

Which stains visualize H pylori

Answer 32

H&E, Warthin-Starry stain, Giemsa, Toluidine blue and using IHC

Question 33

lysochromes stains (cytology)

Answer 33

lysochrome dyes are those which stain fat tissue/lipids. Such as sudan III or IV and oil red O

Question 34

What are the Pap and H&E made to do

Answer 34

promote contrast between adjacent cells as well as their organelles. They allow the viewer to obtain information regarding the cell’s maturity, morphology and metabolic state