FIXATION Flashcards
TYPES OF FIXATION
PHYSICAL METHODS FREEZE DRYING AND FREEZE SUBSTITUTION CHEMICAL FIXATION DEHYDRANT COAGULANT FIXATIVES NON-COAGULANT CROSS LINKING FIXATIVES
PHYSICAL METHODS
HEAT FIXATION
MICROWAVE FIXATION
is useful in studying soluble materials and small molecules
tissues are cut very thinly, immersed in liquid nitrogen, and the water is removed by vacuum chamber at temperatures of _
Tissue can be post-fixed with formaldehyde vapor
FREEZE DRYING
-40°C
__, tissues are immersed in cold (-40°C) fixatives such as _ or _, which slowly remove water by dissolution of ice crystals and the proteins are not denatured.
Bringing the temperature gradually to __ will complete the fixation process
In FREEZE SUBSTITUTION
acetone or alcohol
4°C
utilizes organic and non-organic solutions to maintain adequate morphological preservation
CHEMICAL FIXATION
acts by coagulating proteins and thus making them insoluble
COAGULANT FIXATIVES
DEHYDRANT COAGULANT FIXATIVES
Alcohols and acetone
NON-COAGULANT CROSS LINKING FIXATIVES 10
Aldehydes Gluteraldehyde Formaldehyde Chloral hydrate Glyoxal Metal salts Mercuric chloride Zinc chloride Metallic compounds Osmium tetroxide
Coagulates nucleic acids but does not fix or precipitate proteins
ACETIC ACID
Can penetrate hydrophobic domains of proteins and anion produced (C-COO-) reacts with charged amine groups
This action thus precipitates proteins and extracts nucleic acids
TRICHLOROACETIC ACID(Cl3CCOOH)
Dissolves in water to form a weak acid solution (pH 2.0)
It forms salts with basic groups of proteins, causing proteins to coagulate
PICRIC ACID
is the most commonly used fixative used in diagnostic pathology
Formaldehyde in its 10% buffered form (NBF)
is a vapor then when completely dissolved in water forms a solution containing 37-40% formaldehyde (This aquaeous solution is called __)
Pure formaldehyde
FORMALIN
Commonly used buffers are 5
At an acidic pH, unbuffered formalin produces hemoglobin degradation by-products of brown-black, insoluble crystalline birefringent pigment – formation of undesirable pigments are dependent on how the solution becomes very acidic (pigment forms at a ph__)
phosphates, cacodylate, bricarbonate, TRIS, and acetate
pH of 5.7
Additives include 4
Addition on no-electrolyte solutions like 3 also improve fixation
Calcium chloride,
potassium thiocyanate,
ammonium sulfate
potassium dihydrogen phosphates
sucrose, dextran, and detergents
Fixation is at least 2 weeks
BRAIN
RENAL BIOPSIES
!!
Carson’s modified Millonig’s fixative or 2% buffered gluteraldehyde (pH 7.3)
MERCURIC FIXATIVES
iodine treatment followed by sodium thiosulfate.
Zenker’s fluid Helly’s fluid Schaudinn’s solution Ohlmacher’s solution Carnoy-Lebrun Fixative B5 fixative
DICHROMATE FIXATIVES
Tissue should be washed after fixation and transferred to 70% ethanol. Failure to do so may cause pigments to precipitate.
Orth’s solution
Regaud’s solution
Miller’s solution
PICRIC ACID FIXATIVES
Bouin’s fluid
Hollande’s solution
DEHYDRANT FIXATIVES 3-4 hours of fixation
Absolute ethanol 95% ethanol 70-95% ethanol 100% methanol 100% acetone
is useful for touch preparations and smears especially blood smears
In general most alcohol fixatives should be prepared not more than 1-2 days before use.
METHANOL
is useful to open membranes of intact cells to facilitate entrance of large molecules.
Cold acetone
produces extensive shrinkage and hardening and causes microscopic distortions. Used in immunohistochemistry, enzyme studies and detection of rabies.
Acetone
