Fixation Flashcards
10% aqueous formalin
○ Formaldehyde, 37-40%
○ DI water
Hypotonic fixative that may produce formalin pigment (acidic)
10% formalin saline
○ Formaldehyde, 37-40%
○ Sodium chloride
○ DI water
Isotonic fixative that may produce formalin pigment
Calcium formalin
○ Formaldehyde, 37-40%
○ Calcium chloride
○ DI water
Recommended for the fixation and preservation of phospholipids. The Ca ions prevent the phospholipids from taking up water and distorting.
Formalin ammonium bromide
○ Formaldehyde, 37-40%
○ Ammonium bromide
○ DI water
Recommended for central nervous system, especially in conjunction with the Cajal astrocyte procedure.
It is acidic, lyses RBC, and cause nuclei to give a direct positive Feulgen reaction)
Acetate formalin
○ Formaldehyde 37-40%
○ Sodium acetate
○ DI water
10% neutralized formalin
○ Formaldehyde, 37-40%
○ DI water
○ Calcium or magnesium carbonate
Not recommended as a fixative because the solution becomes acidic after withdrawal from the storage bottle
10% neutral-buffered formalin (NBF)
○ Formaldehyde, 37-40%
○ DI water
○ Sodium phosphate, monobasic
○ Sodium phosphate, dibasic
Most widely used for routine formalin fixation. pH of 6.8-7.2 and is hypotonic.
Modified Millonig formalin
○ Formaldehyde, 37-40%
○ DI water
○ Sodium phosphate, monobasic
- Sodium hydroxide
Isotonic with a pH of 7.2-7.4. Allows electron microscopy on stored tissue
Define the purpose of fixation.
○ Prevent autolysis and putrefaction from occurring so cells and tissue can be viewed as they appeared in their living state.
○ Insolubilize cellular components and
○ Bring out differences in refractive indexes and to increase the visibility of, or the contrast between, different tissue element
Define: Autolysis
Destruction or digestion of tissues and cells by the enzymes normally present in cells
Define: Fixation
The stabilization of protein
Define: Artifact
A structure or substance not normally present but produced by some external force or action (e.g. mercury pigment, tissue floaters, knife lines, air bubbles)
Define: Pigment
A heterogenous group of substances that contain enough natural color to be visible without any further staining (e.g. melanin - a brownish-black pigment found normally in skin, hair and eyes / hematin - brownish black pigment found in blood rich tissues fixed in acidic formalin solution)
Define: Denaturation
To change the nature of.
○ Fixatives denature proteins by changing the soluble (liquid) contents of the cell into insoluble substances so that those substances are not lost during the subsequent processing steps.
○ This change can be caused by either chemical (fixative solutions) or physical (heat, desiccation) means.
○ Denaturation causes the protein molecule to unfold and the internal bonds to become disrupted
Define: Nonaqueous fixative
Solutions that do not use water
Define: Coagulating fixative
This fixative transforms the protein of the cytoplasm into fine mesh which does not interfere with light microscopy but not used in electron microscopy. They separate proteins from water as coagulum
e.g. zinc salts, mercuric chloride, curric sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid.
Define: Additive fixative
Chemically link, or add themselves, to the tissue and change it with this action.
e.g. mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc sulfate or chloride
Define: Non additive fixative
Acts on tissue without chemically combining with it. Dissociates bound water molecules from tissue protein groups, eliminating the solubility of the protein and causing it to precipitate. Excessive removal of bound water results in shrinkage and hardening. Predominately organic compounds like acetone and the alcohols.
Define: Hypertonic
A solution that has a higher concentration of solutes (particles) compared to the inside of a cell. This causes water molecules to move out of the cell, leading to cell shrinkage.
Define: Isotonic
Fluids into which normal animal cells can be placed without causing either swelling or shrinkage of the cells. A 0.9% solution of sodium chloride (saline) or a 5% glucose solution is approximately isotonic
Describe the effect of temperature on tissue fixation.
○ Can affect tissue morphology
○ An increase in temperature increases the rate of fixation but also the rate of autolysis and diffusion of cellular elements (up to 45C has little effect on morphology)
○ 0-4C was considered ideal for electron microscopy
Describe the effect of size on tissue fixation.
○ Sections should be less than or equal to 3mm thick, even thinner for short protocols (at no time should a section be so thick that is touches the both the top and bottom of the cassette)
○ Large specimens like segments of the colon or small intestine should be surgically opened before being placed in fixative to reduce autolysis of the epithelium
○ Solid organs like spleen or kidney should be “bread-loafed” as soon as possible and covered with adequate fixative
Describe the effect of volume ration on tissue fixation.
○ Fixative volume should be at least 15-20x greater than the tissue volume
○ 2-way exchange: The diffusion of the fixative into the tissue displaces the free water and serum proteins of the tissue
○ A tissue volume that is too large will compromise the fixative composition and therefore alter the characteristics
Describe the effect of Ischemic time on tissue fixation.
○ Ideally the tissue should be placed in fixative immediately, the more time that elapses between interruption of the blood supply and fixation, the more postmortem changes that can be demonstrated microscopically (enzyme rich tissue like liver, pancreas, and brain are more subject to rapid autolysis
○ Often seen as surface autolysis (degradation or loss of epithelium)
Describe the effect of duration on tissue fixation.
○ Adequate fixation is needed so that the morphology of the tissue is preserved and tissue will not be distorted by the subsequent processing steps
○ Tissue must be removed from certain fixatives, washed if indicated, and then stored in an appropriate storage solution - if allowed to remain in these fixatives for too long, the tissue becomes over hardened and staining may be impaired
Describe the effect of fixative choice on tissue fixation.
○ Specimens for immunofluorescence study or enzyme profile must be frozen without fixation
○ Rarely a particular fixative must be chosen to ensure optimal demonstration of a particular tissue element
○ Some tissue elements cannot be demonstrated if the wrong fixative is chosen
Describe the effect of penetration rate on tissue fixation.
depth (d) to which the fixative penetrates is proportional to the square root of time (t) hours the tissue was in fixative. K being the coefficient of diffusibility d=k√t
○ The Medawar constant (k) value increases as the rate of penetration increases
○ Increased pressure improves formalin penetration
○ The presence of muscle fibers and capillaries as well as increased surface area with crypts can increase penetration rate
○ Order of decreasing penetration rates: formaldehyde > acetic acid > mercuric chloride > methyl alcohol > osmium tetroxide > picric acid
○ Rate of penetration also impacted by heat
Describe the effect of tissue storage on tissue fixation.
Wet tissue storage is important because the wet tissue often will be needed for additional studies
Describe the effect of pH on tissue fixation.
Influences the reactivity of the fixative and ultrastructure preservation
Describe the effect of Osmolality on tissue fixation.
Describe the properties, functions, and actions of Acetone.
Describe the fixation properties, functions, and actions of Alcohols.
Describe the fixation properties, functions, and actions of B-5 solution.
Describe the fixation properties, functions, and actions of Bouin solution.
Describe the fixation properties, functions, and actions of Carnoy & methacarn solution.
Describe the fixation properties, functions, and actions of Davidson solution.
Describe the fixation properties, functions, and actions of Formalin.
Describe the fixation properties, functions, and actions of Aqueous Formalin.
Describe the fixation properties, functions, and actions of Buffered Formalin.
Describe the fixation properties, functions, and actions of Neutralized Formalin.
Describe the fixation properties, functions, and actions of Acetate Formalin.
Describe the fixation properties, functions, and actions of Formalin alcohol.
Describe the fixation properties, functions, and actions of Calcium Formalin.
Describe the fixation properties, functions, and actions of Formalin ammonium bromide.
Describe the fixation properties, functions, and actions of Gendre solution.
Describe the fixation properties, functions, and actions of Glutaraldehyde.
Describe the fixation properties, functions, and actions of Glyoxal.
Describe the fixation properties, functions, and actions of Helly solution.
Describe the fixation properties, functions, and actions of Hollande solution.
Describe the fixation properties, functions, and actions of Mercuric chloride.
Describe the fixation properties, functions, and actions of Orth solution.
Describe the fixation properties, functions, and actions of Osmium tetroxide.
Describe the fixation properties, functions, and actions of Paraformaldehyde.
Describe the fixation properties, functions, and actions of Potassium dichromate.
Describe the fixation properties, functions, and actions of Zamboni solution.
Describe the fixation properties, functions, and actions of Zenker solution.
Describe the fixation properties, functions, and actions of Zinc Formalin.
Identify the chemicals in and any special indication for B-5 fixative.
Identify the chemicals in and any special indication for Bouin solution.
Identify the chemicals in and any special indication for Carnoy & methacarn solution.
Identify the chemicals in and any special indication for Davidson solution.
Identify the chemicals in and any special indication for Gendre solution.
Identify the chemicals in and any special indication for Helly solution.
Identify the chemicals in and any special indication for Hollande solution.
Identify the chemicals in and any special indication for Orth solution.
Identify the chemicals in and any special indication for Zamboni solution.
Identify the chemicals in and any special indication for Zenker solution.
Identify which fixatives require post-fixation washing & identify the preferred washing agent.
Identify the fixation pigments and the conditions under which the pigment may be formed.
Identify which fixation pigments can be prevented and which of the fixation pigments can be removed, and how.
Explain the difference between buffered and neutralized formalin.
State how paraformaldehyde differs from formaldehyde.
Describe the difference between formalin & formaldehyde.
Identify the percentage and volume of formaldehyde in 1L of a 10% formalin solution.
List 2 methods of fixation other than using chemical reagents.
Identify the preferred method of fixation (or lack of fixation) for Enzyme histochemistry.
Identify the preferred method of fixation (or lack of fixation) for Immunofluorescence.
Identify the preferred method of fixation (or lack of fixation) for Skeletal muscle cross-striation (nonimmunohistochemical staining).
Identify the preferred method of fixation (or lack of fixation) for Pheochromocytomas.
Identify the preferred method of fixation (or lack of fixation) for Urates.
Identify the preferred method of fixation (or lack of fixation) for Immunohistochemical methods.
Identify the preferred method of fixation (or lack of fixation) for trichrome stains.
Identify which fixative reagents are protein coagulants.
Identify which fixative reagents are protein noncoagulants.
Identify which fixative reagents are additive fixatives.
Identify which fixative reagents are nonadditive fixatives
Identify the site or group with which the additive reagent reacts.
Describe the effect of acetic acid on erythrocytes & collagen.
Describe the action of zinc on fixation.
State 2 major problems associated with fixation.
Autolysis & incomplete fixation.
Identify 3 corrective actions for autolysis.
○ Place specimens in fixative solution as soon as possible, ensuring the volume is 15-20x that of the tissue
○ Open uterus specimens upon receipt so the fixative can immediately come into contact with the endometrium
○ Open and pin open gastrointestinal tract specimens upon receipt so that the fixative can immediately come into contact with the mucosal surface
○ Slice any organ resection, breast, or large tissue specimens into thin slices & placing in a large volume of fixative
○ Bisect lymph nodes if appropriate & place in fixative
Identify 3 corrective actions for incomplete fixation.
○ Increase the time allowed in fixative solution. Some problems may be noted even after 24 hours of fixation in 10% NBF
○ Change to another fixative such as zinc formalin, which still requires several hours for complete fixation. Glyoxal should also be considered because it is an extremely rapid fixative
○ Place formalin alcohol in the first 3 stages of the processing cycle. This will decrease fixation time, and also begin dehydration
○ Ensure that the gross sections are thin enough for good reagent penetration, and that the amount of fixative is 15-20x that of the tissue
○ Ensure the formalin solution is not depleted because of overuse, change the solution frequently
○ Do not pack cassettes tightly in the processing holders
○ Use agitation of cassettes in fixative holding solutions during or after gross examination
List 3 criteria for determining the quality of fixation for ultrastructural studies.
Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Osmium tetroxide.
Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Aldehydes.
Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Zamboni solution.
Identify the hallmarks of good fixation.
○ Nuclei with various crisp chromatin patterns & a crisp blue nuclear membrane; nuclei should not show smudginess, bubbling, or fading
○ There should not be any cell shrinkage or artifactual spaces between individual cells
○ Cell cytoplasm should be well preserved & should stain well with eosin
