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Terms in this set (97)
Destruction or digestion of tissues and cells by the enzymes normally present in the cells.
The stabilization of protein
A structure or substance not normally present but produced by some external force or action (i.e air bubbles, knife lines, tissue floaters, mercury pigment)
A heterogenous group of substances that contain enough natural color to be visible without any further staining
Ethyl/Methly Alcohol (Flammables)
1. Allows solution to penetrate or gain entry into the interior of the tissue.
1. Chemically combine with the protein molecules to changed them.
*The protein is made insoluble by the addition of the fixative and then becomes immobilized, remaining at is original location in the cell.
2. Change Tertiary structure of the protein.
3. Chemically link/add themselves to tissue
Cells shrink by releasing fluid
Fluids into which normal animal cells can be placed without causing either swelling or shrinkage of the cells.
Purpose of Fixation
is to kill the tissue so that the postmortem activities of:
pturefaction (bacterial attack)
autolysis (enzyme attack)
The purpose of fixation is to:
* 1. Penetrate rapidly to prevent post-mortem changes.
* 2. Coagulate cell contents into insoluble substances.
* 3. Protect tissue against shrinkage and distortion during dehydration, embedding and sectioning.
* 4. Allow cell parts to become selectively and clearly visible by means of dyes and improved refractive indices.
Helly Fixative contains
Tissue is fixed in order to:
stop enzymatic action
The volume of fixative should exceed the volume of the tissue by:
10 to 20 times
Tissue must be washed in running water after fixation in:
Precipitate left in tissues that have been fixed in solutions containing mercuric chloride may be removed by immersion in:
Commercial stock formaldehyde solutions contain:
37% to 405 formaldehyde
Formalin pigment is most likely to form in tissues if the pH of the formalin solution is AT or BELOW :
Formalin pigment may be removed from tissue by:
Alcoholic picric acid
10% formalin is the same as:
For best results when using formalin as a routine fixative, it must be made:
The ratio of stock solution to acid in Zenker Fixative is:
20 parts stock to 1 part acetic acid
Which of the following tissue changes will occur if acetic acid is used alone as a fixative?
A good fixative for CNS tissue to be stained with silver or gold techniques is:
Formalin Ammonium Bromide
After fixing tissue in Bouin solution, the excess picric acid is frequently removed by washing in:
50% to 70% alcohol
Fats are usually preserved best if the tissue is fixed in:
When alcohol is used as the primary fixative, one should expect:
excessive tissue shrinkage
Generally, an INCREASE in the temperature of the fixative solution:
Increases the speed of fixation
may cause tissue to become overhardened
Fixatives containing chromate salts usually require:
washing in water
Substitution of alcohol as the diluting solution for formaldehyde results in:
better preservation of glycogen
Zinc salts are added to some formalin fixatives to:
provide superior nuclear detail
Tissue fixed in gluataraldehyde is not satisfactory for:
the PAS reaction
will leave the tissue protein uncoagulated
To prevent polymerization of formaldehyde, ________is added to the commercial stock solutions
Polymerized formadelhyde is knows as:
Formaldehyde reacts with protein side chains by combining with the:
Formaldehye cross-links proteins by reacting with________
Example of Nonadditive fixative
Potassium dichormate increases availability of which of the following groups for binding dyes?
is an excellent nuclear fixative
rapidly but fixes slowly
Fixation in Helly solution will preserve erythrocytes
while fixation in Zenker solution will not
Any fixative containing mercuric salts will leave a deposit of
crystals in the tissue
B-5 fixative contains:* done
Sodium acetate (anhydrous)
B-5 stock solution
Bouin Fixative contains:*done
Formaldehyde, 37% to 40%
Picric Acid,saturated aqueous solution (1.2%)
Carnoy Fixative contains:?
10% Neutral Buffered Formalin contains:
Sodium Phosphate, Dibasic
Sodium Phosphate, Monobasic
Gendre Fixative contains:*done
95% Alcohol saturated with picric acid
Helly Fixative contains:
Hollande Fixative contains:*done
Formaldehyde, 37% to 40%
Orth Fixative contains*done
just before use, add
Formaldehyde, 37% to 40%
Zamboni Fixative contains:*done
Picric Acid,saturated aqueous (double-filtered)
Zenker Fixative contains:
Preferred Method of Fixation (or lack of fixation) for:
1. Connective Tissue:
2. Electron Microscopy:
3. Enzyme Histochemistry on muscle
Frozen section (no fixation)
5. Phosphotungstic acid hematoxylin (PTAH)
6. Urate Crystal
1. is recommended for lymph nodes
2. Sections must be treated with Iodine
1.Can be removed with Lugol's Iodine
2. is deposited in tissue during fixation
Acid Hematin (Formalin) Pigment
1. Can be prevented by using a neutral pH
2. Can be removed with alcoholic picric acid
3. is deposited in tissue during fixation
Zenker Fluid & Helly Fluid similarities:
2. must be washed out with running water
preserves erythrocytes in bone
1. May lyse erythrocytes
2. Excess fixative should be removed before processing
Describe the difference between the terms Formalin and Formaldehyde
1. Baker refers to the 37% to 40% solution as "formalin" but bottles obtained from manufacturers are labeled "formaldehyde"
2. Commercial solutions, or stock solutions, are 37% to 40% formaldehyde but are considered to be 100% formalin.
To prepare a 10% formalin solution
1. Dilute one part of the stock solution with nine parts of water.
------Result: the solution is 10% formalin
or 3.7% to 4% formaldehyde
1. has a strong tendency to polymerize to dimers and trimers.
2. only a small part of the formaldehyde is present as a monomer in 37% to 40% solutions, but the monomer predominates in 10% solutions.
-----it is not recommended fixative because the solution becomes acidic after withdrawal from the storage bottle.
Compare and Contrast Zenker and Helley Fixatives
Zenker and Helley:
1. different solutions but both has the mercury present
-----Zenker has the acetic acid
-----Helley has the formaldehyde
List two methods of fixation other than using chemical reagents
Identify any reagents that have associated safety hazards and state the hazard and any special precautions required.
toxic by inhalation of the dust and ingestion
considered a carcinogen
it is corrosive to skin and mucuous
must be tracked and collected for isposal in the same way as mercury
Function of Fixative
1. Kill the tissue so that postmortem activities of decay, putrefaction and autolysis is prevented.
2. maintain proper relationship between cells and extracellular substances
Both contains Formaldehyde
Acid Hematin (formalin) pigment
is deposited in tissue during fixation
a. contains mercuric chloride and potassium dichromate
b. must be washed out with running water.
will lyse erythrocytes because of the acetic acid present but the acid makes it better nuclear fixative
may be used to fix and decalcify needle biopsy specimens of bone marrow, but it may dissolve iron.
will preserve the erythrocytes
-----it is recommended for routine formalin fixation b/c:
1. It has pH 6.8
2. Hypotonic in the buffer ions present (apprx 165 mOsm)
1. a highly polymeric form of formaldehyde, may be deposited as a white powder in concentrated solutions.
2. is used in EM:
a. because they contain no additives
b. used for the preparation of fixative solutions because it yields a pure formaldehyde solutions.
Zenker and Helly Stock Solution*done
Sodium Sulfate (optional)
Identify which fixatives require postfixation washing, and identify the preferred washing agent
Identify the fixation pigments and the conditions under which the pigment may be formed
identify which of the fixation pigments can be prevented and which of the fixation pigments can be removed
for fixation pigments that can be removed, state the method of removal; for fixation pigments that can be prevented, state the method of prevention
Explain the difference between buffered and neutralized formalin
State how paraformaldehyde differs from formaldehyde
Describe the difference between the terms formalin and formaldehyde
Identify the percentage and volume of formaldehyde in 1,000 mL of a 10% formalin solution
Compare and contrast Zenker and Helly Fixatives
List 2 methods of fixation other than using chemical reagents
Identify the preferred method of fixation (or lack of fixation for:
a. enzyme histochemistry
c. skeletal muscle cross-striations (nonimmunohistochemical staining)
e. electron microscopy
g. immunohistochemical methods
h. tissue for trichrome staines
Identify which fixative reagents are protein coagulants and which are noncoagulants
Identify which fixative reagents are additive fixatives and which are nonadditive
If the reagent is an additive compound, identify the site or group with which the reagent reacts (if known)
Describe the effect of acetic acid on erythrocytes and collagen
Identify any reagents that have associated safety hazards and state the hazard and any special precautions required
Describe the action of zinc in fixation
Give the 2 major problems associated with fixation, and identify at least 3 corrective actions for each
Fixation:*frm Htl prep
a. Process of hardening & preserving tissue specimens
b. foundation of all good histological preparation
c. stabilization of proteins
5 Functions fixatives must have: *from HTL prep
1. Prevent post mortem changes of:
autolysis: dissolving of cells die to enzymatic action
putrefaction: breakdown of tissue by bacterial action
2. Preserve cell constituents in as life like manner as possible
3. convert semi-fluid constituency to an irreversible semi-solid constintuency
4. protect by hardening the tissue so it can be further process
5. aid in the visual differentiation of tissues by applying dyes and chemicals
General Information of fixatives: *from HTL prep
1. Important to place in fixative quickly after removal
2. choice of fixative determined by purpose:
exp: Bouin's for connective tissue, Trichrome stains
3. Ideal size of tissues:
3-4 mm thickness,
2 cm square
EM+ 1-2 mm
4. Volume of fixative:
15 to 20 greater than the volume of the specimen
5. Follow the time requirements for each fixative
6. 10% NBF
can mail specimens in it
allows a wide spectrum of special stains
7. Post fixatrion or mandating:
if ideal fix is not sued at first, slides can be post fixed or mordant in required fixative
8. Picric acid (Bouin's, Gender's)
is the only reagent that is fixative and a dye
must be washed in water or 50-80% ETOH
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