simple fixatives Flashcards
acetic acid (7)
generally used for compound fixation bc it balances shrinkage
non coagulant
swells non nuclear proteins
penetrates rapidly
preserves chromosomes, dissolves golgi apparatus and mitochondria
destroys RBCs ( will affect iron stains)
does NOT fix lipids or carbohydrates
safety for acetic acid
store in flammables cabinet not with other acids
store at room temp
store away from strong oxidizers, strong alkalis, nitric acid
concentrated acid safety
store in acid cabinet
pour in fume hood
acid carrier
add acid to water
use heavy rubber gloves
formaldehyde
types: NBF, paraformaldehyde, formalin solution
neutral buffered formalin is most common fixative used
non-coagulant, additive
colorless gas derived from methanol
37-40% full strength
diluted to 10% for use ( formalin)
methanol is added to prevent polymerization
cross links proteins
reacts with amino acids
reacts with NH2 and alters eosin staining
lipids preserved but not insoluble
penetrates quickly, fixes slowly ( 48 hrs@ rm temp )
less shrinkage than all
hardens more than all except alcohol or acetone
cheap, stable
allows more staining than other fixatives
formalin without buffer
without buffer formalin may cause pigment ( known as acid hemtin)
- acidity cause by reaction with O2 in air leads to formation of formic acid, formic acid causes pigment id pHis 6 or lower in the presence of blood
black/brown microcrystalline pigment
- found in blood forming tissue
- bi-fringement
- avoid pH below 6
- remove with alcoholic picric acid, OR 1%potassium or ammonium hydroxide in 80% alcohol
formaldehyde safety
carcinogen
eye and skin irritant
sensitizer
toxic by ingestion
waste disposal considerations
neutral buffered formalin
ph: 7.2-7.4
blood and mucous slow fixation
penetrates 1mm/ hr
fixation equals squared distance of penetration
ex. 5mm from center to periphery would be 5x5 = 25 hrs of fixation
paraformaldehyde
highly polymeric form of formaldehyde
heated and made alkaline for use in EM*
- produces a pure formaldehyde solution
- too difficult for routine use *
formalin solution ( formulas)
formalin ( formal) saline
calcium formalin ( phospholipids )
formal ammonium bromide ( CNS)
- good for cajal nerve stain but gives positive schiff reaction
acetate formalin ( phospholipids)
neutralized formalin ( Ca or Mg carbonate)
neutral buffered formalin *
modified millonig formalin (EM)
alcoholic formalin ( speed )
gluteraldehyde
cross links
dialdehyde
leaves a free aldehyde group ***
- false pos for periodic acid schiff ( PAS)
penetrates slowly and poorly ***
fixes as it pentrates
2 hr fixation for EM - place in buffer to avoid over hardening***
glyoxol
most recent addition to fixatives
smallest dialdehyde
- largely replaced formaldehyde in textile industry
less toxin than formalin
cross linking my be prevented
fixed after 4-6 hrs ( very fast )*
no smudgy nuclei or distorted staining
iron may be decreased - false neg iron stain *
helicobacter pylori staining is unsatisfactory***
RBCs and eosinophilic granules
-possible false neg stains
glyoxal safety
eye and skin irritant
nitrile gloves, goggles and splash proof apron
may be disposed in drain ( money saving)
mercuric chloride
sublime or dichloride of mercury
enhances staining
amorphous brown pigment cannot be prevented
can be removed using iodine followed by sodium thiosulfate ( hypo) ***
birefringent
label pigment removal reagents as “ halogen waste with mercury “ when discarding
mercuric chloride safety
very toxic
affects CNS, or kidney with tiny exposure
bad for environment
osmium tetroxide
post-fixation for EM specimens
additive
non- coagulant
*** preserves lipids and colors them black
penetrates only a few layers
expensive
