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Fixation

Fixation in the histology lab.

QuestionAnswer
Fixative Alters tissue by stabilizing proteins so that they are resistant to further changes.
With additive fixation... The disruption enables the protein to combine chemically with a fixative molecule; making the protein insoluble/
With non additive fixation...(Alcohol, acetone) Denaturation causes the protein to become less capable of maintaining an intimate relationship with water and to become more reactive, but the fixative molecule does not combine with protein.
Putrefaction Bacterial attack
Autolysis Enzyme attack
In tissue that fails to stain, what is one possible reason for it? Autolysis
Tissues that are rich in enzymes: (Prone to autolysis) Liver, pancreas, and brain.
Fixatives kill _ & _ Bacteria & molds
_ can act as a link between stains and tissue. Fixatives
Microwaves are a form of nonionizing radiation
Additive reagents Mercuric Chloride, Chromium Trioxide, Picric Acid, Formaldehyde, Glutaraldehyde, Glyoxal, Osmium Tetroxide, and Zinc Sulfate or Chloride.
Nonadditive fixatives Acetone and the alcohols.
Heavy Metal Cations (+) Chromium, Osmium, Mercury
Anionic Negative charge
Cationic Positive charge
Coagulation Mesh ball (penetrates easily)
Non Coagulent Jello, doesn't allow anything to penetrate it easily.
Coagulent Fixatives Zinc salts, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid.
Non coagulent Fixatives Formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, potassium dichromate, and acetic acid.
After using a non coagulant fixative this type of embedding medium should be avoided due to its lack of ability to penetrate. Paraffin (Plastics is an example of something that could be used)
Fixation Temp for specimens used for Electron Microscopy 0-4 degrees Celsius
Size of specimens should be less than or equal to _mm thick. 3
Fixative should be _-_ times greater than the tissue volume. 15-20
Formalin should have at least _to_ hours to fix before completely being processed. 6-8
Specimens to be fixed for HER2 testing should be fixed in 10%NBF for how long? Minimum of 6 hours and a maximum of 72
Prolonged sating of HER2 testing could have what effect? False negative results.
HER2 testing is used to help identify... Invasive breast cancer
Tissue must be removed from what fixatives? Gultaraldehyde, Helly, Zenker, and Bouins. (If tissue stays in these fixative then the tissue will over harden and staining will be impaired.)
When is no fixation preferred? When performing immunoflourescence or an enzyme profile. Then the tissue is frozen.
When staining muscle cross-striations are to be stained with PTAH (phosphotungstic acid-hematoxyllin) what fixative should be used? Zenker or Bouin
What Fixative should be used when performing a Trichrome stain? Bouins
Mordant used in the Trichrome stain Bouins
Chromaffin granules are found where in the body? Adrenal gland
Chromaffin granules are helpful in identifying what? Pheochromocytomas
Chromaffin granules should be fixed in what? Orth or another primary dichromate fixative
Urate crystals should be fixed in what? Absolute Alcohol
Formalin penetrates _ but continues to cross link proteins for a long time after the penetration is complete. Fast
Fixative ingredients in order of decreasing penetration Formaldehyde, Acetic Acid, Mercuric Chloride, Methyl Alcohol, Osmium Tetroxide, and Picric Acid.
When the specimen is stored in 10%NBF and additional IHC stains are known to be needed in the future then how should the tissue be stored? 70% Alcohol, to prevent crosslinking
Osmolarity The number of particles in a solution.
Isotonic Equal state nothing happens with the cell no exchange occurs
Hypotonic Solution Cell Swells; solution drawn into the cell
Hypertonic Solution Cell shrinks; solution is drawn out of the cell
Kidney biopsies that need to be sent out for testing are placed in what fixative? Michels
What fixatives are preferred for nucleic acids? Acetic alcohol and Carnoy
Lipids are fixed by? Osmium Tetroxide and Chromic Acid
Penetrates fast and leaves tissue soft Acetic Acid
Fixes nuclei and precipitates DNA Acetic Acid
Makes tissue swell Acetic Acid
Lyses red blood cells Acetic Acid
Formaldehyde is what kind of fixative? Non coagulant and additive
Formaldehyde fixes_ but penetrates _ Slowly but penetrates fast
Formalin pigment can be seen by using polarization; what color does it appear as? Red
Types of Formalin fixation 1. microcrystalline dark brown pigment (Formed when acid aqueous solution of formaldehyde acts on tissues rich in blood) 2. Black acid hematin (forms when pH drops below 6)
Formalin pigment is removed by.... Alcoholic picric acid or alkaline alcohol
Formaldehyde solutions are _ but the formaldehyde molecule is not osmatically active . Hypertonic
10% Aqueous Formalin Hypotonic
10% Formalin Saline Isotonic
Formalin Ammonium Bromide Recommended for Cajal Staining, lyses red blood cells, and allows nuclei to give a direct positive Feulgen reaction due to hydrolysis.
Modified Millonig Formalin This is an isotonic dual purpose fixative. Allows tissue to be stored to be used later for electron microscopy. Harder to cut paraffin embedded tissue.
This fixative should not be used when performing a PAS stain. Glutaraldehyde
If tissue for a PAS stain was fixed in Glutaraldehyde the results will be... A false positive
Fixes at the rate it penetrates Glutaraldehyde
Typically used to fix specimens for Electron Microscopy Glutaraldehyde
Over hardens tissue and has poor penetration Glutaraldehyde
Can harden tissue excessively and produce shrinkage Mercuric Chloride
Mercury pigment is removed by Iodine followed by sodium sulfate
Mercury pigment is Bifringent
Preserves lipids Osmium Tetroxide
Coagulent but leaves DNA soluble Picric Acid
Causes extreme shrinkage Picric Acid
Extremely explosive when dry Picric Acid
_ must be completely removed or else all cellular structures will eventually be obliterated. Picric Acid
All reagents should be tightly capped because why? Many of them will absorb water or lose water through evaporation
Tissue is soft Potassium Dichromate
Chromate pigment can be removed by 1% hydrochloric acid and 70% alcohol for 30 minutes.
Typically used for lymph node and bone marrow tissues B-5
Mercuric Chloride Sodium Acetate Formaldehyde B-5
Used for hematopoetic and lymphoreticular tissue B-5
Must be treated for the removal of mercury B-5
Bouins Picric Acid Acetic Acid Formaldehyde
Lyses RBCs Bouins
Iron and calcium deposits are typically dissolved Bouins
Best fixative for the Trichrome stain Bouins
Yellow color from the Bouin fixative must be removed with 50-70% alcohol, or 70% alcohol saturated with lithium carbonate.
Gendre 95% alcohol with picric acid Acetic Acid Formaldehyde
Excellent for the preservation of some carbohydrates, especially glycogen. Gendre
Excess picric acid can be removed with... 80% alcohol
Hollande Copper Acetate Picric Acid Formaldehyde Acetic Acid
Will decalcify small specimens of bone. Hollande
Cupric acetate stabilizes _ membranes and the _ of esinophils and endocrine cells. (Lysis is less severe) RBC membranes and the granules of esinophils and endocrine cells
This fixative must be washed out before the specimen is placed in a phosphate buffered formalin solution of the tissue processor, because if present the solution will form an insoluble phosphate precipitate. Hollande
Formaldehyde Glutaraldehyde Monobasic sodium phosphate Sodium hydroxide Formaldehyde Glutaraldehyde
Store at 4 degrees Celsius Formaldehyde Glutaraldehyde
Stable for 3 months when stored at 4 degrees Celsius Formaldehyde Glutaraldehyde
Zenker Mercuric chloride Potassium dichromate acetic acid
Helly Mercuric chloride Potassium dichromate Formaldehyde
If formaldehyde is not added immediately before use, the solution will darken and become turbid on standing. Helly
Used to fix and decalcify needle biopsy specimens of bone marrow, but can dissolve iron. Zenker
Fixative recommended for PTAH with the exception of silver staining Zenker
Orth Potassium dichromate Sodium sulfate Formaldehyde
Preferred fixative for demonstration of chromatin granules in the cytoplasm of adrenal cells Orth
Demonstration of granules are important in the diagnosis of... Pheochromocytoma
Granules are typically colored _ to _ with chromate Orange to brown
Zamboni Paraformaldehyde Picric Acid NaH2PO4 Na2HPO4
Stable fixative Zamboni
Zinc Formalin Could potentially replace NBF
Antigenicity is enhanced and nuclear detail improved. Alcoholic zinc chloride formalin
Which is preferred Zinc Chloride or Zinc Sulfate Zinc sulfate because it is considered a moderate health risk.
Acetone, Alcohol, Carnoy, Clarke Nonaqueous fixatives
Used as a fixative for brain when a rabies diagnosis is suspected. Acetone
OSHA TWA of 1,000ppm and a National Institute for Occupational Safety and Health TWA of 250ppm Acetone
Used as a fixative for touch preps and blood smears Methyl alcohol
Used to preserve water soluble tissue components Ethyl alchol
Water soluble tissue components Glycogen and urate crystals
Preserves pigment, dissolves fat, and over hardens and shrinks tissue Ethyl alcohol
TWA is 1,000ppm for _ alcohol and 200ppm for _ alcohol ethyl and methyl
Carnoy Ethyl Alcohol Chloroform Acetic Acid
Clarke Absolute Alcohol Acetic Acid
Great fixative for paraffin Clarke
Michel medium is recommended for _ biopsies but not _ biopsies. Kidney but not for muscle.
Michel transport medium Anhydrous citric acid Ammonium Sulfate (MOST) N-ethylmalemide Magnesium sulfate
Discard after 6 months Michel
PBS Potassium phosphate dibasic Sodium phosphate, monobasic Sodium chloride
Primary fixatives for ultrastructural studies include Osmium tetroxide (formaldehyde and glutaraldehyde), and buffered PAF (Zamboni) solutions.
Specimen must be removed within 2-4 hours, preferably less Osmium tetroxide
Specimens can be left in this type of fixative indefinately primary buffered PAF
Fixative that can be used for both light and electron microscopy Primary buffered PAF
How to remove formalin pigment Absolute alcohol with picric acid or 70% alcohol containing ammonium hydroxide
How to remove mercury pigment Treat slides with iodine and then wash with 5% sodium thiosulfate
Created by: Ziek98