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Histology: Fixation
Histology ASCP Fixation information
| Question | Answer |
|---|---|
| What is the definition of a fixative? | A fixative alters tissue by stabilizing the protein so that it is resistant to further changes. |
| Fixative as a process of denaturation | Changes the soluble content of the cell into insoluble substances so that those substances are not lost during subsequent processing steps. |
| What is additive fixation? | A process in which denaturation causes the protein molecule to unfold and the internal bonds to become disrupted. This disruption enables the protein to combine chemically with a fixative molecule, and the protein then becomes insoluble. |
| What is nonadditive fixation? | A process in which denaturation causes the protein to become less capable of maintaining a relationship with water and becomes more reactive, but the fixative molecule does not combine with the protein. |
| Functions of fixatives | 1) Kills tissue to prevent putrefaction and autolysis 2) Maintain the proper relationship between cells and extracellular substances 3) Bring out differences in refractive indexes and to increase the visibility of different tissue elements |
| Actions of fixatives | 1) Inactivates enzymes 2) Kills bacteria 3) Act as mordants 4) Modify tissue for form retention through processing |
| What is denaturation? | A process in which the protein molecule unfolds and the internal bonds become disrupted. |
| Additive fixatives | Chemically link to the tissue to stabilize the protein |
| Nonadditive fixatives | Act on tissue without chemically combining with the tissue to stabilize the proteins. |
| Coagulation | Establishes a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue. |
| What does a noncoagulant fixative do? | Creates a gel that makes penetration by subsequent solutions difficult. |
| List the coagulant fixatives | zinc salts, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, acetone and picric acid. |
| List the noncoagulant fixatives | Formaldehyde, gluataraldehyde, glyoxal, osmium tetroxide and potassium dichromate. |
| What are factors that affect fixation? | Temperature, size, volume ratio, time, penetration, tissue storage, pH, osmolality and choice of fixative. |
| List aqueous fixatives | Acetic acid, formaldehyde, glutaraldehyde, glyoxal, mercuric chloride, osmium tetroxide, picric acid, potassium dichromate and zinc salts. |
| Best fixatives for nucleus | Carnoy and acetic alcohol. |
| Nuclear bubbling | Coalescence of chromatin into strands with intervening clear spaces. |
| Primary structure | Determined by the arrangement of covalent bonds in the amino acid sequence. |
| Secondary structure | Determined by hydrogen bonding between various components of the peptide chain |
| Tertiary structure | Total 3D structure. Hydrogen bonds, ionic bonds, hydrophobic bonds and disulfide bonds are responsible. |
| Best fixatives for lipids | Osmium tetroxide and chromic acid |
| List compound fixatives | B5, Bouin, Gendre, Hollande, Zenker and Helly, Orth, Zamboni, Zinc Formalin and Alcoholic Formalin |
| Does not fix carbohydrates | Acetic Acid, Formaldehyde, Picric Acid |
| Does not fix lipids | Acetic Acid, Picric Acid, Ethanol |
| Penetrates rapidly | Acetic Acid, Formaldehyde, Glyoxal, Acetone, Carnoy, Alcohol, Mercuric Chloride, Potassium Dichromate |
| Increases protein swelling | Acetic Acid |
| Red blood cells are lysed | Acetic Acid, Formalin Ammonium Bromide, Glyoxal, Bouin, Zenker |
| Exposure limit of 10 ppm | Acetic Acid |
| Used in electron microscopy | Paraformaldehyde, Modified Millonig Formalin, Glutaraldehyde, Osmium tetroxide, Zamboni, Carnoy |
| Preserves lipids, but are not made insoluble | Formaldehyde |
| Fixes very slowly | Formaldehyde, Glutaraldehyde |
| Hardens tissue | Formaldehyde, Acetone, Ethanol, Glutaraldehyde, Mercuric Chloride, Carnoy |
| Allows more special stains | Formaldehyde, Glyoxal, B5, Hollande |
| Artifactual pigments | 1) Black acid hematin, birefringent formalin pigment 2) Mercury pigment, birefrigent 3) Chromic pigment |
| Hypertonic | Formaldehyde |
| Hypotonic | 10% Aqueous Formalin, 10% NBF |
| Isotonic | 10% Formalin Saline, Modified Millonig Formalin |
| Recommended fixative for phosphlipid | Calcium formalin |
| Recommended fixative for CNS | Formalin Ammonium Bromide |
| Causes false positive Schiff Reaction | Formalin Ammonium Bromide, Glutaraldehyde |
| Fixes as well as dehydrates | Alcoholic Formalin, Acetone |
| Exposure limit of .75 ppm in 8 hour period | Formaldehyde |
| Exposure limit of 2 ppm over 15 minute period | Formaldehyde |
| Cross-links proteins | Formaldehyde, Glutaraldehyde, Glyoxal, Mercuric Chloride, |
| Penetrates poorly | Glutaraldehyde, Mercuric Chloride, |
| Staining of H. Pylori unsatisfactory | Glyoxal |
| Produces shrinkage | Mercuric chloride, Picric Acid, Ethanol, Potassium Dichromate, Acetone, Carnoy |
| Exposure limit of .002 ppm | Osmium Tetroxide |
| Both a fixative and dye | Picric Acid |
| Recommended fixative for glycogen | Picric Acid,Ethanol, Methanol, Acetone, Gendre, Carnoy |
| Will decalcify tissue | Picric Acid, Bouin, Hollande, Zenker |
| Used for hematopoietic and lymphoreticular tissue | B5 |
| Leaches iron | Bouin, Glyoxal, Zenker |
| Preserves tissue antigenicity | Zinc Salts, B5 |
| Cannot be used with Electron Microscopy | Bouin |
| Recommended for GI | Hollande |
| Cannot be used for silver stains | Zenker, Helly |
| Preserves enzymes | Acetone |
| Fixation for rabies demonstration on brain tissue | Acetone |
| Fixative for touch prep and blood smears | Alcohol |
| Fixative for urate crystals | Alcohol |
| Recommended fixative over Carnoy | Methacarn. Replaces methanol for ethanol, produces less shrinkage and hardening. |
| Mercuric Chloride, Anhydrous Sodium Acetate, Distilled Water, Formaldehyde | B5 |
| Picric Acid, Formaldehyde, Glacial Acetic Acid | Bouin |
| 95% Alcohol saturated with Picric Acid, Formaldehyde, Glacial Acetic Acid | Gendre |
| Copper Acetate, Picric Acid, Formaldehyde, Acetic Acid, Distilled Water | Hollande |
| Mercuric Chloride, Potassium Dichromate, Sodium Sulfate, Distilled Water, Glacial Acetic Acid | Zenker |
| Mercuric Chloride, Potassium Dichromate, Sodium Sulfate, Distilled Water, Formaldehyde | Helly |
| Potassium Dichromate, Sodium Sulfate, Distilled Water, Formaldehyde | Orth |
| Paraformaldehyde, Picric Acid, Sodium Hydroxide, Phosphate Buffer, Distilled Water | Zamboni |
| Absolute Ethyl Alcohol, Chloroform, Glacial Acetic Acid | Carnoy |
| Absolute Methyl Alcohol, Chloroform, Glacial Acetic Acid | Methacarn |
| Not reactive with proteins | Acetic Acid |
| Reactive with nucleic acids | Acetic Acid,Glyoxal, Glutaraldehyde, Mercuric Chloride, Potassium Dichromate, Osmium Tetroxide, Picric Acid |
| Oxidizes carbohydrates to aldehydes | Potassium Dichromate |
| Penetrates slowly | Glutaraldehyde, Potassium Dichromate, Osmium Tetroxide, Picric Acid, Zinc Salts |
| Preservation of nucleoproteins | Acetic Acid |
| Mordant for trichrome | Picric Acid |
| Preserves chromatin and membranes | Glyoxal |
| Avoid Bouin | Feulgen Reaction |
| B5 satisfactory | Methyl Green-Pyronin Y |
| Helly satisfactory | Methyl Green-Pyronin Y |
| Zenker satisfactory | Methyl Green-Pyronin Y, Congo Red |
| Zenker is preferred | May-Grunwald Giemsa, Alcian Blue-PAS-Hematoxylin, Verhoeff, Mallory PTAH, Hotchikiss McManus PAS |
| B5 is preferred | May-Grunwald Giemsa |
| 10% NBF satisfactory | May-Grunwald Giemsa, Congo Red, Masson Trichrome, Mallory PTAH, |
| Bouin is preferred | PAS, Alcian Blue, Masson Trichrome, Hotchkiss-McManus PAS |
| Methyl alcohol is required | Blood smears for PAS |
| Alcoholic Formalin is preferred | PAS D, Muller Mowry Colloidal Iron, Alizarin Red S |
| Absolute alcohol is preferred | PAS D, Best Carmine, Congo Red, Crystal Violet, Turnbull Blue, Von Kossa |
| Carnoy is satisfactory | Best Carmine |
| Bouin is satisfactory | Best Carmine, Congo Red |
| Carnoy is preferred | Muller Mowry Colloidal Iron, Congo Red |
| Avoid Helly | Muller Mowry Colloidal Iron, Aldehyde Fuchsin, Periodic Acid-Methenamine Silver, Steiner and Steiner |
| Avoid Zenker | Muller Mowry Colloidal Iron, Aldehyde Fuchsin, Periodic Acid-Methenamine Silver, Steiner and Steiner |
| Any well fixed tissue | Gomori 1-Step Trichrome, Van Gieson Picric Acid-Acid Fuchsin, Verhoeff, Ziehl-Neelsen |
| Avoid B5 | Periodic Acid-Methenamine Silver, Steiner and Steiner |
| Calcium Formalin preferred | Oil Red O, Sudan Black B |
| Avoid Alcohol | Oil Red O, Sudan Black B, Fontana Masson |
| Avoid Carnoy | Oil Red O, Sudan Black B, Ziehl-Neelsen, Fite, Fontana Masson |
| Avoid Methacarn | Oil Red O, Sudan Black B, Fontana Masson |
| Avoid Gendre | Oil Red O, Sudan Black B, Fontana Masson |
| Alcohol is required | Gomori Methenamine-Silver |
| Avoid Glutarldehyde | Grocott Methenamine Silver |
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