Fixation Flashcards
What are the functions of a fixative
prevent autolysis, stabilize tissue morphology and enhance staining
What are the two major ways that fixatives alter tissue proteins
denaturation and formation of crosslinks
What are the four methods of denaturation
heat, alcohols, acids and heavy metals
What is the most common kind of cross-linking fixatives
aldehydes
What do aldehydes bind to
amino groups as well as lysine, cysteine, serine and threonine residues
How does fixation using oxidizing agents work
poorly understood but it is known that tissues are stabilized by cross-linking reactive groups in close prozimity
What are coagulant fixatives
cause distortion and destruction of fragile cytoplasmic constituents but support proteins. They work quickly
What are examples of coagulant fixatives
alcohols, acetone, acid fixatives and metal salts
What are non-coagulant fixatives
stabilize tissue morphology by forming bridges between adjacent reactive areas within proteins. Slower
What are examples of non-coagulant fixatives
aldehydes and oxidizing reagents
What is an additive
chemically binds with the proteins or other tissue component
What is a non-additive
disrupting tissue structure without becoming chemically incorporated
What are the main factors that affect fixation
temperature, size/thickness/tissue type, time, volume of fixative
What is the minimum fixation time
8 hours
What is 100% formalin
47-40% formaldehyde in water
What is neutral buffered formalin
10% formalin in water
How much formaldehyde is in neutral buffered formalin
3.7-4%
What is paraformaldehyde
a white powder that precipitates out of concentrated formaldehyde solutions
What type of fixative is formalin
additive, non-coagulant, cross-linking
What are the pros and cons of formalin
less shrinkage but hardens tissue
What is a limit of glutaraldehyde
tissue must be very thin
What type of fixative is glutaraldehyde
additive, non-coagulant fixative
When is glutaraldehyde used
for electron microscopy
Why is acetic acid added to fixatives
it preserves nucleoproteins and precipitates DNA, it can lyse RBCs and it swells tissues
What is picric acid
both a fixative and a stain
What type of fixative is picric acid
additive coagulant
Why must picric acid be avoided if DNA/RNA analysis is anticipated
it hydrolyzes nucleic acids
What type of dyes does picric acid enhance
anionic dyes
What must occur after fixation with picric acid
the picric acid must be neutralized
What is ethanol used for in the histo lab
mostly as dehydrating agents
What does fixation with alcohols do to tissues
leaves tissue hard and shrunken
What is ethanol notable for
preserving glycogen and urate crystals
What type of fixative is osmium tetroxide
additive, non-coagulant
What is osmium tetroxide used for
electron microscopy
What makes osmium tetroxide notable
it can fix lipids causing them to appear black
What fixatives are included in bouins solution
formaldehyde, acetic acid and aqueous picric acid
What makes bouins solution ideal
good for trichromes as the picric acid enhances staining, the acetic acid helps counteract shrinkage, and picric acid leaves the block soft and easy to section
Why is it important to wash tissue after fixation with bouins solution
the residual picric acid will digest the section over time
What are the components of B5
mercuric chloride, sodium acetate, formalin and water
What is B5 most often used
hematopeitic and luphoreticular tissues
Why must tissues be stored in 70% alcohol after fixation with B5
it is not tolerant
What is B-plus
a modern version of B5 that uses zinc chloride instead of mercuric chloride
What are the components of Clarke’s fluid
ethanol and glacial acetic acid
What does Clarke’s fluid do
preserves nucleic acids, extracts lipids and maintains microanatomical structures
What is the storage solution after fixation with Clarke’s fluid
95% ethanol
What are the components of modified formalin
formaldehyde and alcohol
What is the benefit of modified formalin
it speeds up tissue fixation and begins dehydration
What does adding zinc sulfate to NBF do
preserves tissue antigenicity with prolonged storage and improves nuclear detail
What is proprietary formalin-free fixative
usually contain an alcohol and other additives, safer solution than formalin
What is melanin
a dark brown, granular pigment often demonstrated using the fontana-masson stain and may be removed using potassium permanganate
What is formalin pigment
amorphous birefringent dark brown or black pigment formed from hemoglobin reacting with formaldehyde in acidic conditions
How to remove formalin pigment from a section
saturated solution of picric acid and alcohol followed by a running water wash
What is mercury pigment
a crystalline birefringent brown pigment
How to correct mercury pigment
use a iodine treatment followed by a sodium thiosulphate treatment
What is picric acid pigment
stains tissue components an intense yellow colour, rinising the tissue helps remove it
What are two examples of exogenous pigments
carbon and tattoo ink
