fixation, decal, dehy, clearing Flashcards
when uses for clearing tissues during the embedding process, is lower in action than xylene but causes less brittleness
chloroform
advantage:
recommended for routine work (6-24hrs)
miscible with absolute alcohol
recommended for rough tissues
suitable for large spx
not inflammable
Disad:
relatively toxic to the liver
clearing is relatively slow
does not make tissues transparent
chloroform
most common chrome -osmium acetic acid fixative used. recommended for nuclear preparation.
Flemming’s solution
composition:
EDTA disodium salt 250gm
distilled water 1750ml
advantage:
permits excellent staining result
produce minimal cell and tissue distortion
Disad
very slow and therefore not reccommended for urgent and routine biopsies
NEUTRAL EDTA
give 6 common dehydrating agents
Alcohol
acetone
dioxane
cellusolve
triethyl phosphate
tetrahydrofuran
its use is reserve for the fixation of cryostat sections or for tissues in which enzymes have to be preserved.
acetone
advantage:
less shrinkage and hardening than ethyl alcohol
excellent for slow processing
miscible with paraffin
disad
odorous
slow acting
long periods of iniltration
low dehydrating power
Butanol (MP: 117.7C)
mercurial fix tissues through an unknown mechanism that increases staining and brightness and gives excellent nuclear detail. however mercurial penetrates poorly and produce tissue shrinkage
metallic fixatives
most commonly used. metallic fixative, frequently used in saturated acqueous solutions of 5-7%
mercuric chloride
is used in 1-2 aqueous solution. it precipitates proteins and adequately preserves carbohydrates
chromic acid
fixation time of regaud’s (muller’s) fluid
12-48hrs
examples of metallic fixatives
mercuric chloride and chromate fixatives
preserves nuclear structure ( e.g. chromosomes). they usually contains glacial acetic acid and their primary component due to its affinity for nuclear chromatin.
ph 4.6 or less
Nuclear fixative
traditionally used for EM. is a good fixative and staining is weak
osmium tetroxide
formula:
mercuric chloride 5gm
potassium dichromate 2.5g
distilled water 100ml
acetic acid, glacial 5ml (to be added just before use)
fixation time: 12-24hrs
Zenker’s solution
formula:
mercuric chloride 5gm
potassium dichromate 2.5g
distilled water 100ml
40% formaldehyde 5ml (to be added)
fixation time: 24-24hrs
Zenker-formol (Helly’s) solution
examples of histochemical fixatives
formol saline 10%
absolute ethyl alcohol
acetone
newcomers fluid
fixation time of formol-corrosive
3-24hrs
thickness of tissue block should be
1.5mm
fixation with formaldehyde is largely complete in _______ hrs
24hrs
the tissue may be stored in cold PBS for how short period of time?
2-3days
decalcification time of formol nitric acid
1-3 days
fastest decalcifying agent
Zenker’s solution
excellent bone decalcifier
neutral EDTA
volatile oil found in citrus peel
limonene
can be dehydrating agent and clearing agent
tetrahydrofuran
not normally utilized as a routine clearing agent but recommended in clearing embryos, insects and very delicate specimen.
aniline oil
recommended for urgent biopsies
benzene
used to remove excess formalin, cosmic acid, and chromates from tissues fixed in Kelly’s, zenkersm and flemming slxn.
tap water
advantage
it is miscible with both absolute alcohol and paraffin
acts fairly rapidly and is recommended for routine purpose
clears overnight
not carcinogenic
disadvantage
slower than xylene and benzene
tends to acidify in a particular filled vessel
more expensive
highly concentrated solutions will emit fumes that are toxic upon prolonged exposure
toluene
advantage:
recommended for routine work (6-24hrs)
miscible with absolute alcohol
reco for tough tissues ( skin fibroid and decalcified tissues)
suitable for lang tissue spx
not flammable
disad
relatively toxic to the liver
chloroform
most widely used fixative for routine histology
10%. neutral buffered formalin
