FIXATIVES Flashcards
For Routine Paraffin Sections, Electron Microscopy,
Histochemistry & Enzyme Studies
10% FORMALIN
Most effective fixative.
10% FORMALIN
Cheap, Readily Available, Easy to Prepare.
● Relatively Stable.
● Compatible with many stains.
● Fumes irritating to nose and eyes.
10% FORMALIN
10% NaCl (sodium chloride) Solution as diluent.
● Fixation of Central Nervous Tissues.
● Post-Mortem Tissues for Histochemical Examinations.
● Fixation time : 24 hrs.(35 degrees) / 48 hrs. (room
temperature).
10% FORMOL SALINE
Preservation and Storage of Surgical, Post-Mortem
and Research Specimens.
4 TO 24 HRS
10% NEUTRAL BUFFERED
- Fast penetration.
- Non-coagulant and additive.
- Prevents alterations during processing.
- Less shrinkage than other fixatives.
- Not osmotically active.
- Hardens tissue better (except alcohol and acetone)
10% NEUTRAL BUFFERED
Mercuric Chloride as diluent.
FORMOL CORROSIVE
Recommended for Routine Post-Mortem Tissues.
Brightens Cytoplasmic and Metachromatic Stains.
Slow penetration.
FORMOL CORROSIVE
polymerized form of formaldehyde, usually
obtained as a fine white powder, which depolymerizes
back to formalin when heated.
PARAFORMALDEHYDE
It is suitable for paraffin embedding and sectioning,
and also for immunocytochemical analysis.
fixed samples can also be stained
for general histology but the degree of fixation is less vigorous than Bouin’s so the quality of the morphology obtained will be less
PARAFORMALDEHYDE
mixture of paraformaldehyde and glutaraldehyde.
● It is suitable for use when preparing samples for light microscopy in resin embedding and sectioning, andfor electron microscop
KARNOVSKY FIXATIVE
This fixative should always be prepared fresh.
KARNOVSKY FIXATIVE
For Routine Light Microscopy, also for Electron Microscopy.
● Preserves cellular structures better.
● Less Irritating to nose and skin.
GLUTARALDEHYDE
small tissue fragments and needle
aspirates.
2.5% SOLUTION
glutaraldehyde larger tissues
4% SOLUTION
Nuclear components are shown in fine detail.
● Excellent Trichrome Staining.
● Preservation of cell detail in Tissue Photography.
MERCURIC CHLORIDE
Pigments removed by treatment with
85-95% Alcoholic Iodine
Permits brilliant staining of nuclear and connective
tissue fibers.
● For Liver, Spleen, Connective Tissue Fibers, and
Nuclei
● Fixation time: 12-24 hrs.
● Lyses RBC and can make tissues brittle.
ZENKERS FLUID
For Pituitary Gland, Bone Marrow and Blood
Containing Organ (e.g. Spleen and Liver).
ZENKERS FORMOL (HELLY’s SOLN)
● Preserves Cytoplasmic Granules
ZENKER-FORMOL (HELLY’s SOLN)
Tumor biopsies of skin.
● Excellent Cytologic Fixative.
● Fixation time: 3-12 hrs.
● Penetrates and fixes tissue rapidly and evenly.
HEIDENHAIN’s SUSA
BONE MARROW BIOSPSIS
B-5 FIXATIVE
Fixation time: 4 – 8 hours
● RAPID FIXATION: 1 1/2-2 hrs
● Unstable.
● Can make tissues brittle.
● Good clear nuclear details.
B-5 FIXATIVE
Preserves Carbohydrates and precipitates all proteins.
May produce sub oxide precipitates.
USUALLY CONSTITUENT OF COMPOUND FIXATIVE
1-2% CHROMIC ACID
LIPIDS AND MITOCHONDRIA
3% POTASSIUM DICHROMATE
Recommended for demonstration of Chromaffin
tissues (phaeochromocytoma), Mitochondria, Mitotic Figures, Golgi Bodies, RBC, and Colloid-containing
tissues(Adrenal glands)
FIXATION TIME: 12-48
PENETRATES WELL
REGAUDS FLUID (MULLER)
Fixation time: 36-72 hours
Recommended for study of Early Degenerative
Processes and Tissue Necrosis
ORTHS FLUID
Recommended for Acid Mucopolysaccharides.
Mucopolysaccharides could be an inborn
error of metabolism.
Fixes connective tissue mucin.
LEAD FIXATIVES
small tissue rapidly.
Excellent for Glycogen Demonstration.
● Suitable for Aniline Stains.
● Causes RBC hemolysis.
1% PICIRC ACID SOLN
Not suitable for frozen sections– causes it to
crumble when cu
1% PICRIC ACID SOLN
recommended for fixation of
embryos and pituitary biopsies.
Preferred fixative for connective tissue staining.
Minimal distortion of
microanatomical structures.
BOUIN SOLN
Minimal distortion of microanatomical structures.
Excellent for soft and delicate tissues.
BOUIN SOLN
Fixation time: 4 – 18 hours
Recommended for gastro-intestinal tract specimens
and fixation of endocrine tissues
HOLLANDES SOLN
Less messy than Bouin’s solution.
Excellent for Glycogen staining.
BRASILS ALCOHOLIC PICRO FORMOL
Overnight tissue fixation by automatic processing
technique may utilize 3-4 changes of Brasil’s fixative
at 1/2 to 2 hours each, succeeded directly by absolute
alcohol.
BRASIL’S ALCOHOLIC PICRO FORMOL FIXATIVE
Rapidly denatures and precipitates proteins
by destroying hydrogen bonds to stabilize
the tertiary structure of proteins.
ALCOHOL FIXATIVES
Recommended for fixing dry and wet smears, blood
smears and bone marrow tissues.
100% METHANOL
Preserves Nucleoproteins and Nucleic Acids for
Histochemistry and Enzyme studies.
Dissolves fat and lipids.
70-100% ETHANOL
May be used as a simple fixative.
Fixes blood, tissue films and smears.
May shrink tissue
70-100% ETHANOL
The most rapid fixative(1-3 hrs only).
● Fixes and dehydrates at the same time.
● Used to fix the brain (fragile specimen)
RBC LYSIS
CARNOYS FLUID
Fixation time: 3-4 hours
has been used on frozen sections
and smears.
CLARKES SOLN
Fixation time: 1 - 6 hours
● Recommended Applications.
● It is sometimes used to fix diagnostic cryostat
sections.
FORMOL ACETIC ALCOHOL
Post-fixation with phenol-formalin
for 6 hours or more can enhance immunoperoxidase
studies.
ALCOHOL FORMALIN
Useful for Sputum analysis as it
coagulates mucus.
GENDRES FLUID
Isopropanol, Propionic Acid, Petroleum Ether, Acetone, Dioxane.
● Acts both as Nuclear and Histochemical Fixative.
● Produces better reaction in Feulgen stain
NEWCOMERS FLUID
Recommended for fixing mucopolysaccharides.
● Fixation time: 12-18 hours at 3°C.
NEWCOMERS FLUID
can react with various side
chains of proteins and other biomolecules, allowing
formation of crosslinks that stabilize tissue structure
but cause extensive denaturation despite preserving
fine cell structure and are used mainly as secondary
fixative.
OXIDIZING AGENTS
Fixes conjugated fats and lipids permanently by
making them insoluble to alcohol and xylene during
dehydration and clearing
6% OSMIUM TETROXIDE
Main Purpose: Fixes materials for Ultrathin Sectioning in Electron Microscopy.
● Drawback: Very Expensive.
● Penetrates poorly.
● Forms black precipitates upon exposure to sunlight.
6% osmium tetroxide
With 1% Chromic Acid as diluent.
● Fixation time: 24-48 hrs
● Recommended for nuclear preparation of such
sections.
● Permanently fixes fat.
● Excellent fixative for nuclear structures
FLEMMING SOLN
Recommended for cytoplasmic structures particularly the Mitochondria
FLEMMING SOLN WITHOUT GLACIAL ACETIC ACID
Sometimes incorporated into compound fixatives.
● It precipitates proteins.
● Softening effect on dense fibrous tissues.
● May be used as a weak decalcifying agent.
● Poor penetration.
TRICHLOROACETIC ACID
Used at ice cold temperature (-5 to 4 °C).
● Recommended for the study of water diffusible
enzymes i.e. Phosphatases and Lipases.
● Used in Fixing Brain Tissues for diagnosis of Rabies.
● Volatile.
● Dissolves fat.
ACETONE
Stable medium for transport of fresh unfixed tissues,
such as renal, skin and oral mucosa biopsies, which
will undergo subsequent frozen section and
immunofluorescence studies.
MICHELS SOLN
Involves Thermal Coagulation of tissue
protein.
■ For Rapid diagnosis;
■ Employed for Frozen Tissue
Sections and Bacteriologic Smears
(gram stain).
HEAT FIXATION
well-known artifact that may be
produced under acid conditions. The pigment may be
eliminated or reduced by fixation in phenol - formalin.
FORMALIN PIGMENT
may be found in surgical specimens
particularly in liver biopsies, associated with an
intense eosinophilic staining at the center of the tissue
in H&E stained sections.
CRUSH ARTIFACT
due to partial coagulation of
partially fixed protein by ethanol or by
incomplete wax impregnation during
subsequent histological processing.
CRUSH ARTIFACT
7 . Tissue can be stored in formalin indefinitely (except for
IHC).
8. Fixative of choice for Immunohistochemistry and
molecular tests.
9. Cheap and stable.
10% NEUTRAL BUFFERED
Chemical constituent
taken into the cell,
forming molecular
complexes and
stabilizing proteins.
Additive
Non coagulant
Chemical constituent
taken into the cell,
forming molecular
complexes and
stabilizing proteins.
Additive
Non coagulant
Fixing agent is not
incorporated into the
tissue
Dehydrating coagulant fixative
Alteration of tissue composition
Non additive
Coagulant
Prevents autolysis and inactivated infectious agents
Allows sectioning of tissue by hardening tissues
Improves cell acidity for special stains
Functions as mordants/accentuators
BENEFITS OF STAINING
Satisfactory fixation occurs between pH
pH 6 and 8
increasing the temperature, increases
the rate of diffusion into the tissue and speed up the rate of chemical reaction between the fixative and tissue elements.
TRUE
Fixation of surgical specimens are done at room
22-25
Temp for electron microscopy
0-4
Nucleic acids do not react with fixatives at room
temperature
TRUE
THICKNESS OF THE SECTION FOR EM
1-2 mm
THICKNESS OF SECTION FOR LM
2 cm
Brain is usually suspended whole in 10% buffered
formalin for 2-3 weeks
TRUE
Tissue thickness is important especially with
non-coagulant fixatives
TRUE
Large specimens like colon resections should be
opened or sectioned at small intervals prior to fixation.
TRUE AT 3-4 mm
The best result is usually obtained using slightly
hypertonic solutions(400-450 mOsm).
TRUE
0.25% Glutaraldehyde - for immunoelectrochemistry
TRUE
Prolonged fixation may cause shrinkage and
hardening of the tissue and may severely inhibit
enzyme activity and immunological reaction.
TRUE
Ideal time to perform fixation is ____
minutes after the interruption of blood
supply.
20-30 minutes
Tissue must be immersed in the fixative for
no longer than ______
60 minutes
Interval between the interruption of blood
supply and the time the tissue is immersed in
the fixative.
cold ischemia time
Time period the tissue is exposed to formalin
or fixative.
fixation time
A commonly quoted rate of penetration for aldehyde fixative is ______ per hour and slows down as it goes deeper in the tissue
2-3mm
_____ times the volume of the tissue to be fixed.
10-25 times
_____ times the volume of the tissue to be fixed.
10-25 times
_____ times the volume of the tissue to be fixed.
10-25 times
tissues should be fixed in a
sufficient volume of solution; generally in a
ratio of 20:1 or at least 10:1 fixative to
specimen.
TRUE
tissues should be fixed in a
sufficient volume of solution; generally in a
ratio of 20:1 or at least 10:1 fixative to
specimen.
TRUE
The maximum effectiveness of fixation is
noted to be 20 times the tissue volume
(1:20).
FIXATIVE:TISSUE VOLUME
TRUE
Fibrous organs take longer than small or loosely
textured tissues such as biopsies or scrapings
TRUE
Fixation can be cut down by using heat, vacuum,
agitation, or microwave.
TRUE
The pH of fixatives does not affect light microscopy.
TRUE
Formalin which is not buffered, is acidic, and has low
pH (pH 4) has little effect on the fine structure of cells
TRUE
At low pH, formalin produces a _____ which
can obscure cellular detail.
DARK PIGMENT
It is necessary to buffer formalin at ______ to prevent
this occurrence
pH 7
The tissue selected for sectioning should be thin
enough to allow penetration by fixative within a
reasonable amount of time.
TRUE
To maintain an adequate fixation time of _____
the tissue size must be _____
fixation time: 4-6 hours
tissue size: 2cm
is used to slow down decomposition if
the tissue that needs to be photographed cannot be
fixed immediately.
REFRIGERATION
is used to slow down decomposition if
the tissue that needs to be photographed cannot be
fixed immediately.
REFRIGERATION
alcohols and acetone
remove lipids and dehydrate the cells, while
precipitating the proteins on the cellular architecture.
Organic solvents
alcohols and acetone
remove lipids and dehydrate the cells, while
precipitating the proteins on the cellular architecture.
Organic solvents
form intermolecular bridges, normally through free
amino groups, thus creating a network of linked
antigens.
cross linking reagents
(paraformaldehyde)
Cross-linkers preserve cell structure better than
organic solvents, but may reduce the antigenicity of
some cell components, and require the addition of a
permeabilization step, to allow access of the antibody
to the specimen.
CROSS LINKERS BETTER THAN ORGANIC
may be used
to preserve phospholipids.
BAKERS FORMAL CALCIUM
