FIXATIVES Flashcards by FRANCHESCA MAUREEN CUISON

For Routine Paraffin Sections, Electron Microscopy,
Histochemistry & Enzyme Studies

10% FORMALIN

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Most effective fixative.

10% FORMALIN

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Cheap, Readily Available, Easy to Prepare.
● Relatively Stable.
● Compatible with many stains.
● Fumes irritating to nose and eyes.

10% FORMALIN

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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

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Preservation and Storage of Surgical, Post-Mortem
and Research Specimens.

4 TO 24 HRS

10% NEUTRAL BUFFERED

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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

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Mercuric Chloride as diluent.

FORMOL CORROSIVE

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Recommended for Routine Post-Mortem Tissues.

Brightens Cytoplasmic and Metachromatic Stains.

Slow penetration.

FORMOL CORROSIVE

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polymerized form of formaldehyde, usually
obtained as a fine white powder, which depolymerizes
back to formalin when heated.

PARAFORMALDEHYDE

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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

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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

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This fixative should always be prepared fresh.

KARNOVSKY FIXATIVE

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For Routine Light Microscopy, also for Electron Microscopy.

● Preserves cellular structures better.

● Less Irritating to nose and skin.

GLUTARALDEHYDE

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small tissue fragments and needle
aspirates.

2.5% SOLUTION

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glutaraldehyde larger tissues

4% SOLUTION

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Nuclear components are shown in fine detail.
● Excellent Trichrome Staining.
● Preservation of cell detail in Tissue Photography.

MERCURIC CHLORIDE

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Pigments removed by treatment with

85-95% Alcoholic Iodine

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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

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For Pituitary Gland, Bone Marrow and Blood
Containing Organ (e.g. Spleen and Liver).

ZENKERS FORMOL (HELLY’s SOLN)

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● Preserves Cytoplasmic Granules

ZENKER-FORMOL (HELLY’s SOLN)

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Tumor biopsies of skin.
● Excellent Cytologic Fixative.
● Fixation time: 3-12 hrs.
● Penetrates and fixes tissue rapidly and evenly.

HEIDENHAIN’s SUSA

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BONE MARROW BIOSPSIS

B-5 FIXATIVE

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Fixation time: 4 – 8 hours

● RAPID FIXATION: 1 1/2-2 hrs
● Unstable.
● Can make tissues brittle.
● Good clear nuclear details.

B-5 FIXATIVE

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Preserves Carbohydrates and precipitates all proteins.
May produce sub oxide precipitates.
USUALLY CONSTITUENT OF COMPOUND FIXATIVE

1-2% CHROMIC ACID

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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

FIXATIVES Flashcards

(102 cards)