Microtechniques 1 - Staining Flashcards
4 different staining mechanisms?
- Acid-base reactions
- Metal impregnation e.g. gold
- Elective solubility e.g. fat stains
- Vital dyes e.g. trypan blue
What is the CHROMOGEN?
Whole dye molecule
What is the CHROMOPHORE?
Part responsible for colour
NO2 or azo -N=N-
What is the AUXOCHROME?
Part that binds to tissue - ionisable to either acid or base.
COO- or NH3+
What is the MODIFIER?
Part responsible for altering shade or tone of colour
-CH^3 (a methyl - the more of them the bluer something is)
What is special about picric acid?
It is a fixative as well as a dye. Some of its side groups act as chromophores and auxochromes.
Basic tissue components = _____philic
Acidophilic e.g. haemoglobin
Acid tissue components = _____philic
Basophilic e.g. DNA, RNA
What are the 2 ways in which a mordant can act?
- Bind dye to tissue permanently e.g. sulphates
2. Stabilise dye and render it insoluble in the tissue e.g. picric acid, mercury
What does a typical mordant look like?
They are usually metal ions e.g. Al3+ or Fe3+
____ - ____ - _____ = LAKE
DYE, MORDANT, TISSUE
Difference between PROGRESSIVE and REGRESSIVE staining?
P: stain for long enough until desired colour is obtained
R: overstain and then remove dye until desired colour is obtained - this gradual removal is known as DIFFERENTIATION
What is the ability of a dye to stain different parts of a tissue different colours called?
What is an example of a dye that does this?
METACHROMASIA
e.g. toluidine blue
What 7 things affect how well a dye binds to tissue?
- The charge (ionisation) of the dye and tissue
- Porosity of the tissue
- Size of the dye particle
- Concentration of dye solution
- Solvent - either water or alcohol
- Temperature
- Time
What does CI stand for? Who came up with it? What are 2 benefits of it?
Colour Index, US Commission of Standardisation of Biological Stains, allows standardisation and consistency between batches.
2 varieties of MOUNTING MEDIA?
- Natural resins e.g. Canada balsam
- Synthetic e.g. xylene - good because RI is v close to glass and we want the same RI
Good MM should be: durable, won’t corrode stain, adhesive
What are the 2 ways to prepare calcified tissues?
- DECALCIFICATION: using acid e.g. HCl (faster) or chelating agents e.g. EDTA (binds Ca2+ preferentially, slower, gentler) - removes calcified components but soft tissue elements retained
- GROUND SECTIONS: section sliced with a saw and then polished (ground) until thin enough to transmit light - calcified components remain but soft tissue elements damaged
