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Fixation
| Question | Answer |
|---|---|
| destruction/digestion of tissue and cells by enzymes | autolysis |
| stabilize proteins | fixation |
| substance/structure not normally present, produced by some external action | artifact |
| heterogenous group of substances, contain enough color that they are visible w/o staining | pigment |
| are primarily acetone and ethyl/methyl alcohol (flammables) | Nonaqeous fixative |
| cloudiness, flocculation, and clot formation stages in coagulation | coagulation |
| chemically link/add themselves to tissue | additive fixative |
| solution that causes cells to swell | hypotonic |
| fluids into which normal animal cells can be placed w/o causing swelling or shrinkage of cells | isotonic |
| allows solution to penetrate or gain entry into the interior of the tissue | coagulating fixatives |
| formaldehyde, osmium tetroxide, potassium dichromate and acetic acid. | noncoagulant fixatives |
| do not allow good penetration by reagents | noncoagulant fixatives |
| Alcohol, zinc salts, mercuric chloride, chromium trioxide and picric acid | coagulating fixatives |
| stabilize and denature proteins | heat |
| mercuric chloride, chromium trioxide, picric acid, formaldehyde, gluteraldehyde, osmium tetroxide, and zinc sulfate or chloride | common additive reagents |
| act on tissue w/o combining with it | acetone and alchol |
| alters tissue stabilize proteins, so that its resistant to further change | fixative |
| kill the tissue so that postmortem activities of decay, putrefaction and autolysis is prevented | Function of fixative |
| maintain proper relationship between cells and extracellular substances | Function of fixative |
| will fail to stain | severely autolysed tissue |
| must change solube contents of a cell into insoluble so that those substances are not lost during processing | Fixative |
| causes proteins to be less capable of maintaining an intimate relationship w/water and become more reactive, but fixative molecule doesnot combine w/t protein | Denaturation |
| Render enzymes inactive, (brain, pancreas, liver) kill bacteria and molds | Action of fixatives |
| Make tissue receptive to dyes, Modify tissue for the maximum retention of form through subsequent processing | Action of fixative |
| Stabilize tissue elements so subsequent procedures will have minimal effect | Action of fixative |
| Physical and Chemical | Methods of Fixation |
| Heat and dessication | Physical |
| Microwave - heat tissue in saline | Heat method |
| Easily overheat, loss of enzyme activity and atingenicity, false locatlization of nuceleic acid, lysis of RBC's | Disadvantage of heat method |
| Increase spee, less destructive to connective tissue | Advantages of heat method |
| Another physical method of fixing proteins | Dessication |
| Air Drying, Basic use: peripheral blood smears, Wright staining most frequent use for this method | Dessication |
| Method that is rarely used | Dessication |
| Primary method of protein stabilization | Chemical fixation |
| additive vx nonadditive, Coagulant vx noncoagulant, Aqueous vx nonaqueous | Reagents can be classified as |
| elements that affect the quality of fixation | Fixation factors |
| Temperature, Size of tissue, Volume ration | Factors influencing fixation |
| increased temp causes increased autolysis and increased diffusion of cellular elements | Factors influencing fixation |
| Size- Sections should not be more than 3mm thick | Factors influencing fixation |
| 15 to 20 times greater than the tissue volume | Fixative volume |
| Tissue should be placed in fixative immediately after surgical removal | Time |
| tissue underlying epithelium | lamina propria |
| Penetration, tissue storage, pH, osmolality | What to consider when choosing a fixative |
| Number of particles in a solution | Osmolality |
| Begins at Periphery of tissue and proceeds inward | Fixation |
| achieve their full effect on tissue at any particular depth as soon as they have penetrated to that depth at a concentration sufficient to cause coagulation | Coagulant fixatives |
| Affected by heat, but not the concentration of the fixative | Rate of penetration |
| Penetrates faster than any of the common fixatives ingridients | Formaldehyde |
| Stored wet | tissue storage |
| tissue may remain in this solution indefinitely | Neutral buffered formalin |
| Solutions with a pH value above 7.0 are | Alkali (base) |
| Solutions with a pH value below 7.0 are | Acidic |
| Solutions with a pH value of 7.0 are | Neutrals |
| is not important in light microscopy but is for electon microscopy | pH of solution |
| Fixatives used for EM need to be at a physiological | pH 7.2-7.4 |
| most fixatives pH range between | 4-9 |
| pigment may be produced if pH drops below | 4.0 |
| have an osmolality of 340 mOsm | Body fluids |
| is a semipermealbe membrane that allows water molecules to pass through it very readily | Cell membrane |
| passes through the cell membrane toward the most concentrated solution to equalize the concentration on both sides of the membrane | Water |
| Water leaves the cell and the cell shrinks | When the cell is surrounded by a hypertonic solution |
| The cell swells possibly rupturing its membrane (cell cytosol) | When the cell is placed in a hypotonic solution |
| physiological or normal | isotonic |
| Additive vx nonadditive, coagulant vx noncoagulant, aqueous vx nonaqeuous | Fixative Classification |
| chemicaly combine with the protein molecules to changed them. the protein is made insoluble by the addition of the fixative and then becomes immobilitzed, remaining at is original location in the cell. | additive fixatives |
| change tertirary structure of the protein | additive fixatives |
| do not combine with protein | nonadditive fixatives |
| they act on the proteins to coagulate them and precipitate them, but do not change their chemical structures | nonadditive fixatives |
| The primary mechanism by whic these fixatives act is to dissociate bound water molecules from tissue protein groups | nonadditve fixatives |
| Mercuric chloride | additive |
| chromium tetroxide | additive |
| picric acid | additive |
| formaldehyde | additive |
| gluteraldehyde | additive |
| osmium tetroxide | additive |
| zinc sulfate | additive |
| zinc chloride | additive |
| methyl alcohol | nonadditive |
| ethyl alcohol | nonadditive |
| acetone | nonadditive |
| transformation of a liquid or a solid into a semi-solid or solid mass | coagulant fixative |
| forms a semisolid network of molecules that allows other preservative solutions to penetrate the tissue deeply | coagulant fixative |
| establishes a network in the tissue that allows solutions to readily penetrate or enter most freely | Coagulation |
| act by creating a gel that makes penetration by a subsequent solution difficult | noncoagulant fixative |
| forms a solid gel which does not allow for easy penetration of solutions and are not used for easy penetration of solutions and are not used for paraffin embedding | noncoagulant fixative |
| Alcohol | coagulant |
| zinc salts | coagulant |
| mercuric chloride | coagulant |
| chromium trioxide | coagulant |
| picric acid | coagulant |
| Formaldehyde | noncoagulant |
| gluteraldehyde | noncoagulant |
| osmium tetroxide | noncoagulant |
| potassium dichromate | noncoagulant |
| acetic acid | noncoagulant |
| Water based | Aqueous fixatives |
| most commonly used | Aqueous fixatives |
| formaldehyde | Aqueous fixative |
| acetic acid | Aqueous fixative |
| Picric acid | Aqueous fixative |
| organic compound known as simple aldehyde | Formaldehyde |
| occurs naturally as a colorless gas | Formaldehyde |
| used interchangeably | Formaldehyde and formalin |
| 37-40% and are considered 100%formalin | Commercial grade formaldehyde |
| 100% formalin is diluted to 10% formalin, which means the concentration of formaldehyde is 3.7-4% | Formalin fixation |
| Formalin | additive fixative |
| it reacts primarily with the amino groups on the amino acids of proteins to form bonds which cross link protein chains together | Formalin |
| nonacuagulating fixative forming a gel which makes the proteins insoluble and keeps them in place and are able to with stand further processing | formalin |
| causes less shrinkage to tissue | Formaldehyde |
| It is hypertonic it is not osmotaically active | Formaldehyde |
| preserves morphological detail so that tissue is life-like | Formaldehyde |
| penetrates tissue rapidly | Formaldehyde |
| Fixes tissue slowly up to 7days | Formaldehyde |
| cross linking proteins because it is an additve fixative | Formaldehyde |
| Chemically stable and kept at room temperature | Formaldehyde |
| tissue can be stored long term without distorting the tissue | Formaldehyde |
| hardens tissue more than other fixatives except ethanol and acetone | Formaldehyde |
| can be used as a simple aqueous solution or w/ addition of sodium chloride to achieve the correct osmolality | Formaldehyde |
| Buffers are added to maintain neutrality (pH of 6.8)and to prevent pigments/percipitates from forming | Formaldehyde |
| organic acid | Acetic acid |
| undiluted is called glacial acetic acid | Acetic acid |
| Diluted is known as vinegar and is used in pickling | Acetic acid |
| leaves tissue very soft | Acetic acid |
| it doesnt fix lipids or carbohydrates | Acetic acid |
| lyses red blood cells | Acetic acid |
| Hypertonic effect on cells | Acetic acid |
| coagulant fixative | Picric acid |
| penetrates tissue well | Picric acid |
| gives tissue a soft consistency | Picric acid |
| Causes extreme shrinkage | Picric acid |
| Not a good fixative for nucleic acids as it leaves DNA solube | Picric acid |
| not the fixative of choice for nucelic acid stains | Picric acid |
| Tissue must have picric acid washed out in 50% ethanol before processing so distortion or obliteration of almost all cellular structures does not occur | Picric acid |
| Toxic and explosive | Picric acid |
| Other than formaldehyde or gluteraldehyde most fixative solutions are combined so that the disadvantage of one component will be counterbalanced by an advantage or disadvantage of another | Compound or combined fixatives |
| hypertonic/hypotonic balance | Compound or combined fixatives |
| mercuric chloride, sodium acetate and formaldehyde | B-5 fixative |
| used to fix hematopoietic and lymphoreticular tissues | B-5 fixative |
| gives beautiful nuclear detail | B-5 fixative |
| wash in iodine the sodium thiosulfate to remove mercury pigments before processing | B-5 fixative |
| store wet tissue in 70% alcohol | B-5 fixative |
| great for immunofluorescence and immunohistochemistry | B-5 fixative |
| contains picric acid, acetic acid and formaldehyde | Bouin solution |
| Good for nuclear preservation | Bouin solution |
| Used for gastrointestinal and endocrine bx | Bouin solution |
| Not used for EM studies or nucleic acid demonstration | Bouin solution |
| Wash in 50-70% alcohol to remove yellow color of fixative | Bouin solution |
| Not for long term storage, place tissue in 70-80% alcohol | Bouin solution |
| NO water | nonaqueous fixatives |
| coagulating, nonadditve fixative | nonaqueous fixatives |
| are used only when the desired tissue components are destroyed or dissolved by aqueous fixatives | nonaqueous fixatives |
| overharden tissue | nonaqueous fixatives |
| shrink tissue | nonaqueous fixatives |
| acetone | nonaqeuous fixatives |
| ethyl/methyl alcohol | nonaqueous fixatives |
| used for enzyme studies especially acid and alkaline phosphatase | Acetone |
| used to fix brain tissue for diagnosis of rabies | Acetone |
| frequently used on frozen sections used for cell surface antigens by IHC | Acetone |
| Touch preps and blood smears | Methyl alcohol |
| Preserves water soluble tissue components like glycogen and urate crystals that are deposited in gout | Ethyl alcohol |
| preserves pigments, dissolves fat, overhardens and shrinks tissue | Ethyl alcohol |
| color deposits which may be found in or on tissue | Pigments |
| Result from chemical processing | Artifact pigments |
| black acid hematain pigment | Formalin pigment |
| brown crystalline pigments forms in tissue rich in blood when the pH is below 6.0 | Formalin pigment |
| it is removed by treating slides with a solution of alcohol saturated w/picric acid or 70% alcohol w/ammonium hydroxide | Formalin pigment can be removed |
| these pigments can be confused w/relative pigments or microorganisms | Formalin pigment |
| found w/mercury containing fixatives (B-5) were not removed prior to processing | Mercury pigment |
| red in color | Mercury pigment |
| Treat w/Gram's or Lugol's iodine followed by sodium thiosulfate | Mercury pigment can be removed |
| nucleus, proteins, lipids, carbohydrates | cells that react w/fixatives |
| Dna, Rna and attached proteins are found in the | Nucleus |
| acetic and carnoy solution | fixatives for nucleic acids |
| produces nuclear bubbling where nuclei show coalescence of the chromatin into strands with clear spaces | Formalin fixation |
| have a primary, secondary and tertiary structure which determine the size and shape of protein molecule | Proteins |
| is determined by the sequence of the amino acids which are linked by covalent bonds | Primary protein structures |
| is determined by hydrogen bonding between the amino acids | Secondary protein structures |
| is determined by other types of chemical bonds between the amino acids such as ionic bonds, hydrogen bonds and linkages with sulfur atoms | tertiary protein structures |
| Many fixatives preserves lipids but only two fix them, so that they are not lost in subsequent processing | Lipids |
| two chemicals that fix lipids | Osmium tetroxide and chromic acid |
| some are lost in fixation | Carbohydrates |
| the storage form of glucose | glycogen |
| glycogen the storage form of glucose is thought to be retained by entrapment of fixed proteins | carbohydrates |
| for immunofluorescence study or enzyme profile | specimen must be frozen w/o fixation |
| tissue should be frozen, sectioned and briefly fixed in acetone | Immunohistochemistry T and B cell markers for dx of lymphomas |
| Special stain | choice of fixatives |
| for a trichrome fix in | Bouins |
| for chromaffin granules found in cells of the adrenal gland fix in | Orth's |
| for urate crystrals fix in | Alcohols |
| when muscle cross-striations are to be stained with PTAH use | Zenkers |
| cells shrink by releasing fluid | hypertonic |
| resist extracgtion by most strong acids, water, alcohol or acetone | Formalin pigments |
| highly polymeric form of formaldehyde | Paraformaldehyde |
| strong tendency to polymerize to dimers and trimers | formaldehyde |
| glutaraldehyde (most used in em) and osmium tetroxide | Electron microscopy fixation |
| Zenker | skeletal muscle cross striation (fixation) |
| use Orth solution when you need to demonstrate Chromaffin granules | pheochromocytomas |
| require nonaqeous fixated absolute alcohol | Urate crystals |
| Frozen w/o fixation | immunofluorescence |
| Bouin | tissue for trichrome (muscle) |
| frozen w/o fixation | enzyme histochemistry |
| frozen sectioned and briefly fixed in acetone | immunohistochemistry |
| a cell is a solution that is more concentrated than the cell cytosol | hypertonic |
| NH2 | amino group |
| -COOH | Carboxyl group |
| Chromium, mercury and osmiums | Heavy metals |
| are cations that combine with anion group of proteins | Heavy metals |
| positvely charged | cations |
| negatively charged | anions |
| sulfhydryl, carboxyl and phosphoric | combine w/anions |
| -Sh | sulfhydryl |
| -PO4 | phosphoric acid |
| most rapidly penetrating component of aqueous fixative | water |
| has freezing point of 16.6C | glacial acetic acid |
| prepared in water | aqueous |
| B-5, Bouin, Gendre, Hollande, Zenker/helly, Orth, Zamboni, Zinc formalin | compound fixative |
Created by:
nperez
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