Fixation Flashcards
Define autolysis
Cells self-destruct using their own enzymes released by lysosomes.
Define fixation
Stabilizing cell proteins by making them insoluble. This way, they resist changes, such as autolysis and putrefaction.
Tissue, properly fixed, is preserved in a life-like manner, has improved refractive contrasts, and is more receptive to dyes.
What is fixation artifact?
A substance or tissue alteration from an outside source.
What is a fixation pigment?
An artifact chemically produced by cell component/fixative interaction
Explain the difference between buffered and neutralized formalin.
Formalin can be neutralized (with calcium or magnesium carbonate), but it will still become acidic (pH 5-5.7) if buffering salts are not added. Buffering salts bring the pH to 7.2-7.4.
What is black acid hematin?
Formalin pigment. An artifactual pigment that forms when hemoglobin reacts with formic acid and the solution drops below a pH of 6.0.
Which is formaldehyde’s physical state? Gas, liquid or solid?
Gas (HCHO).
What speeds the process of dissolving formaldehyde gas molecules in water?
Buffering salts.
In what physical state is formaldehyde received in the laboratory?
Liquid (HO-CH₂-OH), a 37%-40% aqueous solution chemically called methylene hydrate.
What is the difference between formaldehyde and formalin?
Formaldehyde is a colorless gas. Formalin is an aqueous solution (technically methylene hydrate).
What percentage formaldehyde is actually in 10% formalin?
3.7% to 4.0%
If a commercial solution labeled 37%-40% formaldehyde is received in the lab, is it really formalin?
Yes. It is considered 100% formalin by histotechs. Pure formaldehyde is technically a gas.
What is the most commonly used fixative in the histology laboratory?
10% Neutral Buffered Formalin
What does NBF stand for?
Neutral Buffered Formalin
Is alcohol a fixative?
Yes alcohol is one of two primary nonaqueous fixatives. The other is Acetone.
What are the 3 types of alcohol used as fixatives?
Ethyl, methyl, and isopropyl
Which alcohol is drinking alcohol?
Ethyl
How does paraformaldehyde differ from formaldehyde?
Formalin polymerizes into paraformaldehyde if left untreated with methyl alcohol.
What is done to methylene hydrate (formalin) to slow down the polymerization into paraformaldehyde?
10% to 14% methanol is added to commercial 37%-40% formaldehyde (which is correctly called methylene hydrate).
Is paraformaldehyde a fixative?
Yes. It is used at about 4% and is preferred to formalin for EM due to the absence of methanol, which tends to distort cell components. Although, Carson states that Modified Millonig Fixative (made with 37%-40% commercial formaldehyde) can be used for both light and electron microscopy.
What is a methylene bridge?
A cross link between aldehydes and nitrogen or proteins. A methylene bridge occurs when collagen is tanned to make leather.
Why do we say formalin penetrates quickly but fixes slowly?
Because it can bind with proteins within 24 hours but takes longer for methylene bridges to form. This is what leads to inadverdent alcohol fixation on the tissue processor.
What does absolute mean when referring to alcohol?
Pure. Less than 1% water.
What is the name for pure acetic acid?
Glacial acetic acid.
Why do we call concentrated acetic acid glacial?
Because it freezes at 16.6°C
True or False-RBC’s are lysed by acetic acid.
True
What are the 2 primary nonaqueous fixatives?
Alcohol and acetone.
What happens if you combine alcohol and acetic acid?
You get the fixative called Clarke Fluid.
What is the chemical definition of conformation?
The arrangement (especially bonding) of atoms in molecules.
What is the chemical definition of denature?
To disrupt the conformation of biomolecules. For example: To denature a protein is to unravel its 3 dimensional shape by breaking hydrogen bonds.
What is cytolysis?
Osmotic lysis, or cytolysis, happens when a cell swells until it bursts due to excess water moving into the cell.
What is the difference between a coagulative fixative and a non-coagulative fixative?
Coagulant fixatives create a mesh-like network which aids penetration of subsequent reagents. (such as alcohol). Non coagulative fixatives create a gel that makes penetration more difficult. (such as formalin).
What is ischemic time?
The time from the interruption of the blood supply to the beginning of fixation.
Name the major factors affecting fixation in general.
Time (both ischemic and duration), temperature, size, volume ratio, penetration, and pH.
Explain how temperature affects fixation and why it can be complicated.
As with other chemical processes, the rate of fixation increases as temperature increases and decreases as temperature falls. Choosing between cold fixation, room temperature fixation, and fixation involving heat can be a difficult decision because, of course, temperature affects autolysis in the same way.
Find the artifactual pigment in this image and name it.
Black acid hematin, or formalin pigment, forms when a specimen containing hemoglobin is fixed in acidic formalin.
How is formalin pigment removed from tissue sections?
By treating with saturated, alcoholic picric acid or 10% ammonia in 70% alcohol.
True or False: If a cell is placed in a HYPERtonic solution, it will shrink.
True. The cell will plasmolyse.
Water will leave the cell to try to equalize.
What is plasmolysis?
The opposite of cytolysis. The protoplasm of the cell shrinks and the cell dies.
How is Osmium Tetraoxide used in histology?
It the oldest fixative for EM. Its use as a fixative for FFPE is limited because it interferes with staining by acidic dyes. However, it does completely fix fat.
