STEP 1: FIXATION (unfinished) Flashcards
used as a mordant to perform special staining
secondary fixative
post-chromatization is a form of secondary fixation where a primarily fixed tissue is placed in aqueous solution of __________________ for 24 hours as a mordant
2.5-3% potassium dichromate
used to wash out chromates, formalin, and osmic acid
tap water
used to wash out excess amount of picric acid
50-70% alcohol
used to wash out excess mercuric fixation
alcoholic iodine
fixative becomes part of the tissue
additive fixation
fixative stability by forming cross-links
additive fixation
fixative is not incorporated into the tissue
non additive fixation
stability by removing bound water attached to H-bonds of certain groups within the protein molecule
non additive fixation
fixation temperature for surgical specimens
room temp
fixation temperature for electron microscopy and … histochemistry
0-4C
fixation temperature for bacteriology and blood films
heat
fixation temperature for urgent biopsies
formalin heated to 60C
fixation temperature for tissues with tuberculosis
formalin at 100C
thickness of section for electron microscopy
1-2 mm^2
thickness of section for for light microscopy
2 cm^2 or no more than 0.4 cm
osmolality that gives best results
slightly hypertonic solutions
isotonic solutions cause…
cell swelling
volume of fixative
should be 10-20 times the volume of tissue
fixation is retarded by…
size and thickness
presence of mucus
presence of fat
presence of blood
cold temperature
fixation is enhanced by…
size and thickness
agitation
choice of fixative and mode of processing is dependent on…
need for immediate examination
tissue structure or component to be studied
type of tissue
staining technique to be applied
type of section to be made
characteristics of a good fixative
cheap
stable
safe to handle
kills cells quickly
inhibits bacterial decomposition and autolysis
permits rapid and even penetration of tissues
harden tissues
isotonic
makes cellular component insoluble to hypotonic solitions
permits subsequent application of many staining procedures
a primary component common in nuclear fixatives
glacial acetic acid
pH of nuclear fixative
4.6 or less
pH of cytoplasmic fixatives
> 4
fixative that must never contain glacial acetic acid
cytoplasmic fixatives
aldehyde fixative
formaldehyde
10% formol-saline
10% neutral buffered formalin of phosphate buffered formalin
formal-corrosive
alcoholic formalin
glutaraldehyde
commercially available solution of formaldehyde
10% formalin
fixation time for formalin
24 hours
formalin concentration unsatisfactory for routine fixation
40% formalin
a “tolerant” fixative
formalin
disadvantage: reduces basophilic and eosinophilic staining of cells
unbuffered formalin
disadvantage: forms abundant brown pigment granules on blood-containing tissues
unbuffered formalin
recommended for central nervous tissues and general post-mortem tissues
10% formol-saline
ideal for most staining techniques
10% formol-saline
disadvantage: metachromatic reaction of amyloid is reduced
10% formol-saline
10% formol-saline is a simple microanatomical fixative made up of…
saturated formaldehyde diluted to 10% with sodium chloride
recommended for preservation and storage of surgical, post mortem and research specimens
10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
best fixative for tissues containing iron pigments and elastic fibers
10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
for routine post-mortem tissues
formal-Corrosive (Formal-Sublimate)
advantage: no need for “washing out”
formal-Corrosive (Formal-Sublimate)
disadvantage: forms mercuric chloride deposits
formal-Corrosive (Formal-Sublimate)
disadvantage: does not allow frozen sections to be made
formal-Corrosive (Formal-Sublimate)
disadvantage: inhibits the determination of the extent of tissue decalcification
formal-Corrosive (Formal-Sublimate)
aka Gendre’s fixative
alcoholic formalin
used to fix sputum
alcoholic formalin fixative
good for preservation of glycogen and for micro-incineration technique
alcoholic formalin fixative
made up of two formaldehyde residues linked by three carbon chains
glutaraldehyde
used for small tissue fragments and needle biopsies
2.5% glutaraldehyde
recommended for larger tissues less than 4mm thick
4% glutaraldehyde
recommended for enzyme histochemistry and electron microscopy
glutaraldehyde
