Fixation & Fixatives Flashcards
Chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein
ADDITIVE FIXATION
Fixing agent is not incorporated into the tissue but alters the tissue to H bonds of
certain groups within the protein molecule
NON-ADDITIVE FIXATION
pH for satisfactory fixation
Between pH 6 and 8
Electron microscopy workouts and histochemistry examination ideal temperature is
0-4°C
Formalin heated to ___ is sometimes used for rapid fixation of very urgent biopsy
specimens
60°C
Formalin at 100°C can be used
to fix tissues with
tuberculosis
The best results are usually obtained using
slightly hypertonic solutions
PRACTICAL CONSIDERATIONS OF FIXATION
-Speed
-Penetration
-Volume
-Duration of fixation
They act as mordants or accentuators to promote
and hasten staining, or they may inhibit certain dyes
in favor of another
Fixatives
Lead Fixatives
- Picric acid
-Acetic acid
-Acetone
-Osmium tretoxide
-Alcohol
permit the microscopic
study of tissue structures without altering the
structural pattern and normal intercellular
relationship of tissues
Microanatomical Fixatives -
- preserve specific parts and
particularly microscopic elements of the cell itself
Cytological Fixatives
preserve nuclear structures
(e.g. chromosomes) in particular
Nuclear Fixatives
Usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatic
Nuclear Fixatives
must never contain glacial acetic acid since it destroys the mitochondria and golgi bodies; pH of more than 4.6
Cytoplasmic Fixatives
preserve chemical
constituents of cells and tissues
Histochemical Fixatives
Phospholipids which contain amino groups are fixed
by
aldehydes
should be used for
demonstrating lipid in tissues by a general lipid stain
Cryostat or frozen sections
Improved ultrastructural demonstration of lipids has
been achieved by post-fixing in
imidazole osmium
tetroxide
Cholesterol may be fixed with ___ for ultrastructural demonstration.
digitonin
can enhance better retention of
glycogen
Celloidin coating
Two aldehyde fixative mixture have been particularly
useful for electron cytochemistry
Karnovsky
paraformaldehyde-glutaraldehyde
Penetrate tissue rapidly, preserves
morphology and enzyme activity at low
concentrations
- Useful for immersion fixation of surgical
biopsies
Acrolein
simple microanatomical fixative made up of saturated formaldehyde (40% by weight volume) diluted to 10% with sodium chloride
10% FORMOL SALINE
fixation of central nervous
tissues and post-mortem tissues for histochemical
examination
10% FORMOL SALINE
Fixation time for 10% Formol Saline
24 hours at 35 °C OR 48 hours at 20-25 °C
recommended for preservation and storage of surgical, post-mortem and research specimens
10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE
BUFFERED FORMALIN
Fixation time for 10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE
BUFFERED FORMALIN
4-24 hours
prevents precipitation of acid formalin pigments; best tissue fixative for tissues containing iron pigments and for elastic fibers that does not stain
well after Susa, Zenker or Chromate fixation
10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE
BUFFERED FORMALIN
Reactivity of myelin to Weigert’s iron hematoxylin stain is reduced; Inert towards lipids, especially neutral fats and
phospholipids
10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE
BUFFERED FORMALIN
recommended for routine post-mortem tissues
FORMAL-CORROSIVE
Forms mercuric chloride deposits; does not allow frozen tissue sections to be made
FORMAL-CORROSIVE
95% Ethyl alcohol saturated with picric acid
➢ Strong formaldehyde solution
Alcoholic Formalin (Gendre’s) FIxative
Good for preservation of glycogen and for micro-incineration technique; Used to fix sputum since it coagulates mucus
Alcoholic Formalin (Gendre’s) FIxative
Produces gross hardening of tissues; Causes partial lysis of RBC; Preservation of iron containing pigments is poor
Alcoholic Formalin (Gendre’s) FIxative
made up of two formaldehyde residues linked by
three carbon chains
Glutaraldehyde
MOST COMMON METALLIC FIXATIVE
MERCURIC CHLORIDE
ROUTINE FIXATIVE OF CHOICE for preservation of cell
detail in tissue photography
MERCURIC CHLORIDE
widely used secondary fixative reacting with a
number of amino acid residues and accompanied by
spectroscopic changes, probably due to histidine
residues
MERCURIC CHLORIDE
Recommended for fixing small pieces of liver, spleen,
connective tissue fibers and nuclei
ZENKER’S FLUID
Recommended for trichrome staining
■ Permits brilliant staining of nuclear and connective
tissue fibers -Compatible with most stains
■ Act as a mordant
ZENKER’S FLUID
Excellent microanatomic fixative for pituitary gland,
bone marrow and blood containing organs such as
spleen and liver
ZENKER-FORMOL (HELLY’S SOLUTION)
Main fixative for tumor biopsies especially the skin.
Excellent cytologic fixative
Heidenhain’s Susa
Used for bone marrow biopsies
B-5 Fixatives
Recommended for demonstration of chromatin,
mitochondria, mitotic figures, Golgi bodies, RBC and
colloid containing tissues
REGAUD’S (MULLER) FLUID
Deteriorates and darkens on standing (must always
be freshly prepared)
REGAUD’S (MULLER) FLUID
Fixation TIme for Orth’s Fluid
36-72 hours
Recommended for early degenerative process and
tissue necrosis; Demonstrates rickettsiae and other bacteria
Orth’s Fluid
Normally used in strong saturated aqueous solutions
➢ This fixative also dyes tissues producing yellow color.
Picric acid
recommended for fixation of embryos and pituitary
biopsies
Bouin’s solution
better and less messy than Bouin’s; excellent fixative for glycogen
BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE
Normally used in conjunction with other fixatives to
form a compound solution
➢ Solidifies at 17 °C
GLACIAL ACETIC ACID
