Fixation Flashcards
what is a fixative?
alters the tissue by stabilizing protein so it is resistant to further changes
what do fixatives do? (4)
prevent autolysis by deactivating enzymes, kills bacteria and molds that can cause putrefaction, makes tissues more receptive to dyes and can act as a mordant, stabilizes cellular structures to resist change during subsequent processing and staining
autolysis
breakdown of tissue due to enzyme activity
putrefaction
breakdown of tissue by bacteria
direction of fixation
outer layer of the tissue moving inwards
appropriate tissue size
3-4mm thick and 2x2cm square
reasons for choosing a fixative
rate of penetration, length of storage, pH for electron microscopy, osmolality for EM, looking for a specific structure that may be adversely affected by certain fixatives
non-coagulant fixative
creates a gel that is difficult for subsequent solutions to penetrate, preferred for electron microscopy due to a more life-like fixation
coagulant fixative
establish a network in tissue that allows solutions to readily penetrate the interior of the tissue
non-additive fixative
act on tissue without chemically combining with it, often by dissociating water molecules from protein
additive fixative
add themselves onto the protein and cause a change
black acid hematin
occurs when formalin is used and the pH drops below 6, especially on tissues rich in blood
can be removed by treating sections with alcoholic picric acid or alkaline alcohol
physical agents of fixation
heat and desiccation
volume of fixation fluid
15 to 20x the volume of tissue
4 factors affecting fixation
time in fixative, temperature of fixative, size of tissue, volume ratio of fixative solution
4 functions of fixatives
kill the tissue, maintain proper relationships between cellular structures, increase contrast between tissue elements via differing refractive indexes, stabilize the tissue physically and chemically
list of simple fixatives
Acetic Acid, formalin, glutaraldehyde, glyoxal, mercuric chloride, osmium tetroxide, picric acid, potassium dichromate, zinc salts
list of compound fixatives
B-5, Bouin, Gendre, Hollande, Zenker, Helly, Orth, Zamboni, Zinc Formalin
Acetic Acid
non coagulant (coagulates) cytoplasm and nucleic acids
does not fix or destroy carbs or lipids
penetrates rapidly
leaves tissue soft
precipitates DNA
Increases protein swelling (disadvantage)
Lyses red blood cells (often counteracted in compound fixatives)
B-5
Good nuclear detail for staining blood forming and lymph tissue
good for many special stains
Bouin
great for soft delicate structures and trichrome stains
acetic acid swelling is countered by picric acid shrinkage
hardening of formaldehyde is countered by soft fixation of picric acid
yellow color is removed with 50-70% EtOH
don’t use for EM because it does not preserve nucleic acids
Gendre Solution
great preservation of carbs, esp glycogen
excess picric acid is removed with 80% EtOH
Hollande Solution
can decalcify small bone specimens
good for GI biopsies
cupric acetate counters the lysis of RBCs and eosinophil granules caused by acetic acid
Zenker
tissues must be treated for mercury pigment with iodine followed by sodium thiosulfate
makes nuclei less basophilic and cytoplasm more acidophilic, increase time in hematoxylin and reduce eosin
good for nuclear chromatin, connective tissue
very toxic and carcinogenic
