Staining Flashcards
Van der waals forces
creates a significant binding force between the dye and protein although individually weak.
Strongest in large molecules as in Elastic fibre stains
hydrogen bonding
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
ionic bonding, types of dyes
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.
Example of anionic/acidic dye and what is stains
eosin, stains erythrocytes, collagen, cytoplasm of many cells
covalent bonds
not veru important in staining. seen in some hsitochemical stains such as Perioidic acid schiff
What is a chromophore
chemical group which makes any organic compound coloured
auxochromes
ionizable groups added onto chromogens to convert them into proper dyes which the tissue can retain
metachromasia and orthochromasia
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
What chemical groups are in acidic and basic dyes and what they dye .
acidic -amino grousp,,mainly proteins in the cytoplasm and connective tissue
basic - used as nuclear stains, carboxylate, phosphtae,sulfate groups.
neutral dye
basic and acidic dyes, with both ions being coloured therefore stain nuclei and cytoplasm. Such as Romanowsky stain: Giemsa.
What does the mordant do
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
what can be used for chemical oxidation of haematoxylin
sodium iodate, potassium permanganate or mercuric oxide
mordent of aluminium haematolxyins
- aluminium potassium sulfate or aluminium ammonium sulfate
examples of Al haematoxylins
Harris’, Mayer’s, Gill’s, Ehrlich’s
examples of iron haematoxylins
Weigert’s, Heidenhain’s, Verhoeff’s
Addition of glacial acetic acid to Harris HAematoxylin
gives more precise and selective staining of nuclei
How different fixatives affect staining with haematoxylin and eosin
formalin fixed tissue take up less eosin. Tixed fixed in solutions containing potassium dichromate tend to take up less haematoxylin
What is histochemistry
The use of special stains to identify the location of chemicals within the tissues - which cannot be seen with H&E
PAS stain componetns
–> 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
Components of MAsson’s Trichrome stain
- 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
How Verhoeff- van Gieson stain Elastin fibres stain works
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
Componetns of congo red and how they work
Pretretment with NaOH to release native internal hydrogen bonds.
NaCl to supress flase negative staining of collagen and elastin. important to carry out control.
Other stains for amyloid
-crystal voilet polychromatic stain stains it purple
-toluidine blue dyes it blue
- sirius red stain stains it pink, green birefringence
- thioflavine reaction
Dealing with sample with prion disease
fixed tissue and slides have to be inactivated by treatment in 96% formic acid for at least 1 hour.
When is visualization of micro organisms in tissue sections useful
- 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.
Stains used for acid fast bacteria
Ziehl Neelson, Wade-Fite, Auramine/Rhodamine
Stains for Spirochetes
Warthin and Starry, Toluidine Blue, Giemsa
Stains for fungi
Grocott-Gomori, PAS
What does acid fast mean
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
how are fungi detected by staining
through oxidation of their rich polysaccharide walls into dialdehydes.
How does grocott’s methenamine silver stain work
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.
Which stains visualize H pylori
H&E, Warthin-Starry stain, Giemsa, Toluidine blue and using IHC
lysochromes stains (cytology)
lysochrome dyes are those which stain fat tissue/lipids. Such as sudan III or IV and oil red O
What are the Pap and H&E made to do
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
