Kaiserling's fluids
Best fixative for museum specimens
Carnoy's fluid
Best fixative for nucleic acid (RNA and DNA)
Formol-Calcium
Best fixative for phospholipids
Zenker's Fluid
Best fixative for phosphotungstic acid-hematoxylin methods
10% NBF
Best fixative for simple fats
Best fixative for trichrome methods for collagen and muscle
Bouin's Fluid
Best fixative for uric acid crystals
Absolute Ethanol
Artifacts of improper fixation
1. Nuclear or cellular membranes are altered or not in tact
2. Mitochondria cristae are displaced
3. Proteins are mobilized
4. Ground substances are unevenly distributed in clumps or destroyed
Characteristics of a coagulant fixative
1. Change the sponge work of proteins into meshes through which paraffin can easily pass
2. Produces more artifacts than non-coagulant fixatives
3. Strengthens protein linkages against breaking during subsequent processing
4. Produces complete fixation as
Actions of Formaldehyde in Helly's fluid
1. Reducing agent
2. Reacts with proteins involving the formation of cross-links between the molecules giving rise to an insoluble product
3. Causes turbidity and the formation of a dark brown precipitate
4. Improves penetration and fixation
Fixation in Zenker's or Helly's fluid requires that the tissue be small enough to ensure complete fixation in
6-8 hours
For the staining of chromaffin cells for the diagnosis of pheocromocytoma, it is necessary to fix the tissues in
Primary Chromate Fixative
Characteristics of Potassium Dichromate
1. Unsuitable for fixation when histochemical techniques are contemplated
2. Used for the fixation of mitochondria
3. Preserves phosphatides
4. Gives fixation to cytoplasm without precipitation
Mercuric Chloride is not used alone as a fixative because
It penetrates poorly and causes excessive tissue shrinkage
Which fixative must be post-treated for the mercuric chloride pigment
Schaudinn's Fluid
Actions of acetic acid in fixatives
1. Exerts a swelling effect on tissues
2. Renders nucleoprotein basophilic
3. Breaks the salt linkages between protein chains
4. Coagulates nucleoprotein
Characteristics of acetic acid as a fixing agent
1. Coagulates nucleoprotein
2. Dissolved out mitochondria and golgi
3. Does not harden the tissue
4. Rapid penetration
Characteristics of the sucrose treatment used in enzyme histochemical studies
1. Sucrose solution contains 30% sucrose and 1% gum acacia
2. It helps membranes stabilize
3. May be stored in the sucrose solution for long periods
4. Frozen sections may be prepared or stained after sucrose treatment
For all histochemical and the majority of histological techniques, it is necessary to bring the pH of formalin to neutral because
Formic Acid is formed in formaldehyde
After fixation with formaldehyde or glutaraldehyde, enzyme activity and preservation of structure depend upon all of the following
1. Concentration and purity of the aldehyde
2. pH
3. Fixation time and temperature
Advantages of 2-3% glutaraldehyde
1. Excellent preservation of all protein
2. Preservation of organelles and inclusion granules
3. Histochemical reactions may be performed on post-fixed specimens
What are the steps in the glutaraldehyde fixation procedure for EM specimens
1. Tissues are initially fixed in 2-4% phosphate buffered glutaraldehyde
2. After glutaraldehyde fixation tissues are rinsed in a phosphate buffer
3. Tissues are post fixed in osmium tetroxide
Steps in glutaraldehyde purification
1. Initially add activated charcoal to glutaraldehyde with pH below 3.5
2. Charcoal-Glut solution allowed to settle for 4hrs at 4C
3. Filter through Whatman filter paper #50
4. Filter 2nd time through millipore filter
Characteristics of Zamboni's PAF
1. Should have a final pH of 7.3
2. Has an Osm of 900mOsm
3. Is alkalinized by NaOH
4. Phosphate buffer is a required component
What fixative is used in lipid histochemistry
Calcium-Formalin
Why does the paraformaldehyde in Karnovsky's (paraformaldehyde-glutaraldehyde) have to be heated to 60C prior to adding the 1M NaOH
To dissociate paraformaldehyde to formaldehyde
Which EM fixative is stable at room temp
Zamboni's PAF
Which of the following is NOT used in carbohydrate fixation?
a. 10% NBF
b. Bouin's fluid
c. Gendre's fluid
d. Schaudinn's fluid
D
What solutions are part of the Kaiserling technique for museum specimens?
Fixative solution, Restoring solution, Preservative solution
What are some characteristics of tissue fixation for enzyme histochemical studies?
-Tissues may be fixed in certain cold fixatives prior to cryostat sectioning
- Fresh tissue may be post-fixed after cryostat sectioning in certain cold fixatives
- Some procedures require sucrose treatment for fixed tissues prior to cryostat sectioning
-
Best fixative for CNS silver impregnation
Formalin Ammonium Bromide
Best fixative for chromaffin cells
Orth's Fluid
Best fixative for cytologic smears
95% EtOH
Best fixative for EM
Zamboni's PAF
Best fixative for mitochondria
Helly's Fluid and Post-Chromatization
For good fixation of specimens for light microscopy, it is recommended that the tissue be no larger than
1mm cubed
Fixative for blood smears
Absolute Methanol
Fixative for bone marrow
Helly's fluid
Fixative for brain tissue for rabies
Cold Acetone
Which cold fixative is not used in IHC studies?
1% Osmium Tetroxide
Which fixative is not recommended for EM?
10% Unbuffered formalin
Which fixative has a mordanting effect on tissue?
Zenker's fluid
Good cytoplasmic fixatives should not contain
Glacial Acetic Acid- because mitochondria and golgi are destroyed by it
What are the constituents of Zamboni's PAF Fixative?
Paraformaldehyde, Picric Acid, Sodium Phosphate
What are the characteristics of Zamboni's PAF fixative?
1. Stabilizes cellular protein
2. Not easily destroyed by tissue fluids
3. May be used without post-osmication
4. Recommended for light and EM
Orth's fluid is the recommended fixative for
1. Chromaffin granules in cytoplasm of adrenal medulla
2. Diagnosis of Pheochromocytoma
3. Ferric Ferricyanide Reduction Test
The major disadvantage in the use of osmium tetroxide as a fixative is
It's tendency to interfere with several staining methods
Helly's fluid is the recommended fixative for
Bone marrow, blood-containing organs, intercalated disc
Characteristics of Helly's fluid
1. Mordant for metachromatic staining
2. Mordant for Geimsa stain
3. Produces and artifact pigment
Characteristics of Zenker's fluid
1. Mordant for metachromatic stains
2. Recommended when tissues are intended for the Feulgen Reaction
3. Fixed tissues must be stored in either 70% alcohol or 10% Formalin
An advantage of fixation in Zenker's fluid is
Zenker fixed tissues stain brilliantly
Characteristics of Carnoy's Fluid
1. Causes considerable shrinkage
2. Destroys or dissolves most cytoplasmic elements
3. Suitable for small tissues
4. Hemolyzes RBC's
Zenker's fluid is the recommended fixative for
Cytoplasmic Inclusions (Viral Inclusions, Negri Bodies)
Advantages of Bouin's fluid
1. has good penetration
2. recommended for soft friable structures
3. excellent for connective tissue stains
4. recommended for the demonstration of glycogen
5. excellent fixative for routine surgical material
Carnoy's fluid is not recommended for the preservation of
Acid Fast Bacilli
Bouin's fluid should not be used for
Tissue to be stained by the Feulgen reaction
Bouin's fluid can be used for
1. Small tissues
2. Tissue intended for connective tissue stains (Particularly trichromes)
3. Routine tissue sections
If tissue prepared with Bouin's fixative is washed in water it will lose some of it's soluble picrates. It should be placed instead in
50% Alcohol
Absolute ethanol is recommended as a fixative for
1. Glycogen
2. Pigments
3. Blood films and smears
Acetone is recommended as a fixative for
1. Fluorescent Antibody techniques
2. Enzymes
3. Brain tissue in the diagnosis of rabies
The function of methanol in commercial formalin is to
Retard the polymerization of formaldehyde into paraformaldehyde
Has the ability to denature protein by coagulation and rapid dehydration, lyses RBC's, Useful for the preservation of larger quantities of glycogen
Absolute ethanol as a fixative
Disadvantages of formalin
1. Noncoagulation of protein
2. Reduces DNA and RNA
3. Interacts with hemaglobin or its products at and acid pH
The chief action of formalin on tissue is
polymerization
10% Formalin adequately fixes tissues at room temp in
6-18hrs
Advantages of formalin
1. Preserves lipids
2. excellent for CNS tissue
3. Compatible with most staining methods
4. sufficiently hardens tissue
5. good for frozen sections
Bone must be fixed prior to decal and may be fixed in
10% NBF
Unbufferd 10% Formalin
Zenker-Formol Variants
Zamboni's PAF refers to
Picric Acid Formaldehyde (PAF)
Tissue must be placed in a suitable fixative as soon as it is removed from the body to
Prevent decomposition of the tissue due to lack of oxygen
Permits more uniform histologic preservation, prevents autolytic changes, and inactivates many enzymes, are all characteristics of
Immediate embalming
Mercuric Chloride pigment is removed from tissue by
Iodine-Sodium Thiosulfate
Which of the following fixatives require removal by water
Zenker's fluid
An unknown pigment in a tissue section which bleaches when washed with a saturated solution of picric acid in alcohol is likely to be
Formalin Pigment
Constituents of 10% NBF are
1. Sodium Phosphate monobasic
2. Sodium Phosphate dibasic
3. 37-40% Formaldehyde
NBF should never be prepared by storage over calcium carbonate or magnesium carbonate, since the fluid drawn off for fixation promptly becomes
acidic
Formalin pigment is generally formed in tissues fixed in formalin when
pH level falls below 6
A stain that exhibits many colors on tissue
polychromatic
Refers to a stain that stains tissue in a predictable way
orthochromatic
To make a 10% formalin solution how many mL of H2O should be added to 300mL of 37-40% Formaldehyde solution?
2700mL
What fixative is used for specimens to be mailed?
10% NBF
A universal fixative used for routine purposes which permits a broad spectrum of staining methods is
10% NBF
10% NBF contains what % of Formaldehyde?
4%
Characteristics of a good fixative
1. makes the tissue more permeable to fluids
2. preserves the tissue in a life-like manner
3. inhibits putrefaction
4. hardens tissue quickly
5. does not shrink/swell tissue
6. renders enzymes inactive
7. does not distort or dissolve tissue parts
Tissue fixation begins
at the periphery of the tissue and proceeds inward
For good tissue fixation, it is recommended that the tissue be no larger than
2cm squared, 3-4mm thick
Improper preservation of tissue can be due to
1. delay in fixation
2. improper storage (prolonged)
3. poor dehydrating, clearing, embedding, and sectioning techniques
Specimens are best stored in
1. 70% EtOH- restores color from formalin fixation and does not effect staining
2. Formalin ok, but causes tissues to gradually lose basophillic staining properties
The chief aim of fixation is the
coagulation (precipitation) of the protoplasmic substances of a cell
Fresh, unfixed tissue can be held safely for a short time by
Wrapping in saline-moistened gauze and placing in a refrigerator
Renders the cells and tissue elements resistant to further changes from the reagents to which they are subjected before microscopic sections can be prepared. Denatures proteins, converts from gel-like state to semi-solid
Coagulation
The rate of fixation varies with fixative and is also dependent upon
temperature
To bring out the refractive index differences in tissue, to harden or affect tissue so that it will not be altered by subsequent processing, to arrest autolysis and putrefaction, and to render cellular contents insoluble are functions of
fixation
A physical agent used in fixation is
heat and dessication
The volume of the fluid being employed should be
15-20X that of the tissue
The breakdown of the tissue due to enzyme activity
autolysis
the breakdown of tissue by bacterial action
putrefaction
The most important step in the preparation of a high quality microscopic slide is
Good Fixation
Functions of Baker's Formalin Calcium Fixative
1. After fixation, the tissues are placed in a dichromate-calcium mordant
2. Fixative for the preservation of phospholipids
3. pH 4.7-4.9
Flemming's solution ingredients
1. 1% chromic acid
2. 2% osmium tetroxide
3. glacial acetic acid
Carnoy's fluid ingredients
1. absolute alcohol
2. chloroform
3. glacial acetic acid
PBS 10% sucrose solution ingredients
1. stock solution
2. distilled water
3. sucrose
PBS Buffer Stock Solution ingredients
1. Potassium phosphate dibasic
2. Sodium phosphate monobasic
3. sodium chloride
Michel Transport Medium Ingredients
1. Anhydrous citric acid
2. ammonium sulfate
3. N-ethlymaleimide
4. magnesium sulfate
5. distilled water
Clark Fluid ingredients
Absolute alcohol and glacial acetic acid
Carnoy Solution Ingredients
1. absolute ethyl alcohol
2. chloroform
3. glacial acetic acid
Alcoholic Zinc Chloride Formalin Ingredients
1. Zinc chloride
2. Distilled water
3. Isopropyl Alcohol, 99%
4. Formaldehyde, 37-40%
Unbuffered Aqueous Zinc Formalin Ingredients
1. Zinc Sulfate
2. Distilled Water
3. Formaldehyde, 37-40%
Aqueous Zinc Formalin Ingredients
1. Zinc Sulfate Heptahydrate
2. Formaldehyde, 37-40%
3. Distilled water
Zamboni Solution Ingredients
1. Paraformaldehyde
2. Picric acid, saturated aqueous
3. sodium phosphate, monobasic
4. sodium phosphate, anhydrous
5. distilled water
Orth Solution Ingredients
1. Potassium Dichromate
2. Sodium Sulfate
3. Distilled water
4. FOrmaldehyde 37-40%
Zenker-Helly Stock Solution Ingredients
1. Mercuric Chloride
2. Potassium Dichromate
3. Sodium Sulfate
4. Distilled water
Zenker working solution
1. Stock Solution
2. Acetic Acid
Helly working solution
1. Stock Solution
2. Formaldehyde 37-40%
Hollande Solution
1. Copper acetate
2. picric acid
3. Formaldehyde, 37-40%
4. Acetic Acid
5. Distilled Water
Gendre Solution
1. Alcohol, 95% saturated with picric acid
2. Formaldehyde, 37-40%
3. Glacial acetic acid
Bouin Solution
1. Picric acid, saturated aqueous
2. Formaldehyde, 37-40%
3. Glacial Acetic Acid
B-5 Fixative Stock Solution
1. Mercuric Chloride
2. Sodium Acetate
3. Distilled Water
B-5 Fixative Working Solution
1. Stock Solution
2. Formaldehyde
Alcoholic Formalin
1. Formaldehyde, 37-40%
2. Distilled Water
3. Ethyl Alcohol, absolute
Modified Millonig Formalin
1. Formaldehyde 37-40%
2. Distilled water
3. Sodium Phosphate, Monobasic
4. Sodium hydroxide
10% NBF
1. Formaldehyde, 37-40%
2. Distilled water
3. Sodium Phosphate, Monobasic
4. Sodium Phosphate, Dibasic
10% Neutralized Formalin
1. Formaldehyde, 37-40%
2. Distilled H20
3. Calcium or Magnesium Carbonate
Acetate Formalin
1. Formaldehyde, 37-40%
2. Sodium Acetate
3. Distilled Water
Formalin Ammonium Bromide
1. Formaldehyde, 37-40%
2. Ammonium Bromide
3. Distilled H2O
Calcium Formalin
1. Formaldehyde, 37-40%
2. Calcium Chloride
3. Distilled water
10% Formalin Saline
1. Formaldehyde, 37-40%
2. Sodium Chloride
3. Distilled Water
10% Aqueous Formalin
1. Formaldehyde, 37-40%
2. Distilled Water
Coagulant
1. Establishes a network in tissue that allows solutions to readily penetrate or gain entry to the tissue
2. Zinc Salts, Mercuric Chloride, Cupric Sulfate, Ethyl and Methyl alcohol, Acetone, Picric acid,
3. Mesh ball (aqueous solutions readily penetrate)
Noncoagulant
1. Creates a gel that makes penetration by subsequent solutions difficult
2. Formaldehyde, glutaraldehyde, glyoxal, Osmium Tetroxide, Potassium Chromate
3. Jello-hard to penetrate
Preferred fixative for nucleic acids
acetic acid and carnoy solution
Preferred fixative for lipids
Osmium tetroxide, chromic acid, calcium formalin