Week 10 Flashcards
What are mechanisms
◦ Processes or techniques by which something works
◦ Sequential
◦ Has beginning and ending
what are the two types of mechanisms
- Physical mechanisms
o Natural phenomenon - Chemical mechanisms
o Artificial phenomenon
What plays a part in the physical M of S
- Adsorption
- Selective solubility
- Porosity
What is Adsorption
what is its capacity provided by
and what forces are responsible for it
◦Combination of tissue and dye is by latent valences of atoms on the surface
Dye (Adsorbed material) + Tissue (Absorbent) - dyes can be removed with water or alcohol- temporary
capacity provided by :
◦ Surface area- larger the area the more place for dye to be and less intense the stain , small area= more intense the stain
◦ Size of pores- dense material = intense stain
◦ Surface polarity - charges on dye , opposite sites attract = lots of binding sites
forces responsible - weaker forces
◦ Van der Waals
◦ Electrostatic forces
difference between Adsorption & Absorption
◦ ADSORPTION: accumulation of gas, liquids, or solutes on the surface of a solid or liquid - can be readily removed
◦ ABSORPTION: transfer of material from one phase to another to form a “mixture”
What is selective solubility
◦ Used by Lysochromes (e.g. Oil Red O) to stain neutral lipids
-dye prefers where it is more soluble and it is more soluble in its own lipid than working solution
◦ Lysochrome dissolves in fat but1 not in its solvent
◦ Cannot use paraffin wax sections; frozen section only
-dye molecules select and choose to be dissolved in lipid
-dye molecules in a working solution of Oil red are unstable because it has a lower alcohol content so they prefer to be in lipid than in the solution where they are soluble.
The molecules would be happier in the stock solution where there is 100% isoprop
What is porosity
◦ Fixed protein precipitates into meshwork that measures void spaces in material
Porosity depends on
◦ tissue densities - densest tissue is least porous (collagen least - RBC most)
◦ tissue porosities
◦ Dye charge
◦ Dye molecular weight
all when tissue is fixed
collagen - least dense and RBCs most dense but both have positive charge. So they have an affinity to negatively charged dyes. The denser the meshwork the more positive charges there are. Therefore dye will enter the collagen first because its less dense but also leaves quicker.
acidophilic tissues - Positive charge
small weight dye larges stay in RBCs longer because the small one stay in the small pores so large ones will only fit in large pore like in collage
What are the chemical M of S
- Non-ionic bonding
- Ionic bonding
- Leuco compounds
- Metachromasia
- Histochemical reactions
- Metallic impregnation
What is non ionic bonding
- no charges involved
-hydrogen bonds
-van der waals forces
collagen and elastin
What is ionic bonding
routine h&e
opposite attract
change of isoelectronic point
if the tissue is
ACIDOPHILIC = POSITIVE
E.g. CYTOPLASM: RBC, MUSCLE, COLLAGEN
ACID DYE- ANIONIC DYE = NEGATIVE
if tissue is
BASOPHILIC = NEGATIVE
E.g. NUCLEI, CHROMATIDS, DNA
BASIC DYE CATIONIC DYE = POSITIVE
Zwitterion form of amino acid when it is pretreated with acid
one has positive charge and other has neg therefore net charge is 0 - IEP
-Adding acid would make the net charge +1 net increase in positive charge
-when acid stain (-) put on this pretreated tissue there would be bright color because of increase in dye binding sites due to increase of positive binding sites on the tissue
-when a basic stain positive charge is put on this tissue there would be a dull color because there are less negative charges on the protein
Zwitterion form of amino acid when it is pretreated with akaline
addition of alkaline would make charge -1
-basic dyes like an alkaline solution
when acid stain (-) is put on the color would be dull because there are less positive charges on the tissue protein
When there is a basic stain (+) applied the color would be bright because there is an increase in number of dye binding sites due to more negative binding sites
What are leuco compounds in staining
◦ Colorless compounds obtained from or converted into colored dye
Dye chromaphore gets reduced
Sulphonation > Schiff reagent
Hydrogenation > carbinol
-Both lose ability to stain because their chromophores were destroyed- leuco compound
The compound loses color - get oxidized by aldehyde group in tissue-chromophoric structure is restored. substance has color again
What is Metachromasia
Chromotrope are tissue components capable of changing the colour of (metachromatic) dyes
META- changing CHROME- colour
tissue (chromotrope can change the color of metachromatic dye) + toluidine blue ->chromotrope stains purple red but most will stain blue orthochromatic
stains purple and not blue because the matric is a chromatrope
What is polymerization
metachromasia takes place when there is polymerization
Basic dye + chromotropes
◦ Chromotropes must be close together and spaced evenly
◦ Chromotropes are basophilic or anionic – negatively charged, metachromatic dyes are cationic – positively charged
◦ Use water as solvent (no alcohol)
◦Presence of water and where the dye molecules will allow Chromotropes to absorb different light from the dye a – while the rest of the tissue pick up the blue colour of the dye.
What are Non-aggregated dye molecules
◦ Chromotropes not spaced evenly will not undergo polymerization
◦ Water was not used as solvent so unevenly spaced
◦ Will still attract metachromatic dyes but will show self colour of the dye - orthochromatic
What is Histochemical staining
◦ Use of non-coloured reagent to produce a non-diffusible substance in tissue-permeants color
-used for Glycogen and Ferric iron -
◦ Colour is imparted at the site of the reaction
-starts with glycogen -liver muscle or F I and a staining solution that does not have a chromophore because they were destroyed but is able to reform and stain
What is the schiff reaction
-◦ Leucofuchsin will react with aldehyde groups to produce a magenta coloured substance
glycogen is oxidized by the periodic acid using dye aldehyde groups then add fuchsin sulpurous acid (Schiff reagent) which reacts with the dye aldehyde in the schiffs protein = produces Magenta colored complex where the targets are located
-dye chromophore has to be destroyed before use
What is the perl prussian blue stain
protein with ferric iron -Hcl ->Ferric iron + potassium ferrocyanide -> ferri ferro cyanide
-target is hemosiderin
staining solution does not have a dye molecule , a tech mixes ferric iron and pos ferrocyanide = perls solution clear pale yellow solution
What is metallic impregnation
◦ Affinity for metals (silver)
Two types of Silver impregnation techniques
◦ ARGYROPHIL
◦ ARGENTAFFIN
◦ Silver nitrate (AgNO3) is used in an unstable form
◦ Silver nitrate (AgNO3) is an alkaline solution
What is the Argyrophil Reaction
◦ Use external reducer to reduce BOUND silver to FREE silver - reticulin fiber
-silver binds to aldehydes in the tissues - with no visible color = because we need an external reducer- like formaldehyde
-bound silver is changed to free silver = black color
–sodium thiosulphate removes unreduced silver to prevent nonspecific blacken on slide
◦ For demonstration of Reticulin fibers
Gordon and Sweet’s Reticulin Stain (GSR)
-collagen, elastin, and reticulin pick up a pink stain in H&E so a special stain needs to be done to differentiate between them
What is the Argentaffin Reaction
◦ Does not need extraneous reducer
-a clear alkaline solution is made
◦ For demonstration of melanin, fungal wall, protozoan wall, basement membrane (phenolic groups)
-phenolic groups will react with the Ag ion in the silver nitrate solution = silver produces black precipitates where the reducing substances are
-silver reduced automatically no formaldehyde needed
Grocott’s Methenamine Silver (GMS)- fungal tissue
What is toning
◦ For both silver impregnation reactions, silver can be enhanced by TONING
◦ Use GOLD CHLORIDE
◦ Gold replaces reduced silver
◦ Mechanisms: ION EXCHANGE and METAL ACTIVITY
What is the difference between direct and indirect staining
on direct it goes right on the connective tissue cell and stains with no help
on indirect, the dye needs tissue AND a mordant for staining
mordant + dye = Lake binds better, stronger reaction aka staining nuclei with aluminum in hematoxylin
mordant - salt of heavy metal with strong affinity for both dye and tissue
Accentuators VS Accelerators
ACCENTUATORS:
-non essential
◦ Act as catalysts in the DYE-TISSUE chemical rxn
◦ e.g. phenol, acetic acid (Ziehl Neelsen stain for TB)
ACCELERATORS:
◦ Substances added to metallic impregnation techniques for the demonstration of CNS
◦ e.g. sodium barbitol (Bielchowsky stain)
Progressive VS Regressive stain
Progressive
wash the slide after some time -Gomori’s aldehyde fuchsin for demonstration of elastin, β cells
Regressive
slide and overstain and differentiate/ decolorize and only what you want to see if stained Hematoxylin and Eosin for routine staining
Decolorization VS Differentiation
DECOLOURIZATION:
◦ Macroscopic removal of visible stain from tissue section
◦ e.g. acid/alcohol (for Ziehl Neelsen) acetone/alcohol (for Gram stain)
DIFFERENTIATION:
◦ Controlled, microscopic removal of stain from certain tissue component
◦ e.g. solvent of the stain, mordants, alkali solutions, oxidizers, other dyes
Acid Differentiation VS Mordant Differentiation
ACID DIFFERENTIATION
◦ Use of acid alcohol for H&E stain
-removed link from cell and tissue
◦ Breaks the LAKE-TISSUE salt linkage
MORDANT DIFFERENTIATION
◦ Use of excess mordant in Verhoeff’s van Gieson (VVG)
◦ Mordant in solution competes with mordant in tissue for the dye molecule
◦ Breaks the DYE-MORDANT bond
◦ Only mordant is attached to tissue
Vital VS Non-vital Staining
VITAL:
◦ Demonstration of living cells using dyes
◦ Supravital –remove cells from body and stain before cell death- RBC
◦ Intravital – inject dye into living organism
NON-VITAL:
◦ Demonstration of dead cells using dyes
Empirical Staining
◦ Trial and error techniques
◦ No documentation of dye chemistry and tissue chemistry
