BOT/MICRO/ZOOL 5364
FIXATIVES
I. Aldehydes
A. Glutaraldehyde (Sabatini, Bensch & Barrnett, 1963)
0 0
C-CH2-CH2-CH2-C MW = 100.12
H H
Advantages:
1. Enzymatic activity partially preserved which permits cytochemical localization following fixation.
2. Penetration rate slightly faster than osmium tetroxide.
3. Exerts some osmotic effect. Effect is dependent on concentration and specimen type. No significant changes observed by changing concentration of glutaraldehyde between 2% and 5%.
Disadvantages:
1. Does not destroy osmotic properties of cells. Osmolarity of fixative must be carefully controlled.
2. Can cause shrinkage of cells at higher concentrations.
3. Tends to polymerize at alkaline pH (above 7.5).
4. Has no staining action.
5. Reacts only slightly with lipids. May cause phospholipids to pass into solution producing myelinic figures upon reaction with osmium tetroxide. Myelinic figures can be prevented by adding 1-3mM CaCl2 to glutaraldehyde fixative, but care must be taken since Ca2+ ions may precipitate proteins.
Commercial Availability:
1. Bulk quantities: solutions (usually 25 or 50%). Contain impurities and polymers of glutaraldehyde. Must be purified.
a. Activated charcoal- 10% (w/v). Shake 1 hr at 4 C. Filter. Repeat 2 or 3 times.
b. Distillation. Collect distillate between 100 and 101 C in 50 ml aliquots. Discard those with pH below 3.4.
c. Vacuum distillation. Distill at 15 mm mercury pressure. Collect distillate at 80-85 C and immediately dilute to 25% with boiling water.
2. Solutions of vacuum distilled glutaraldehyde (usually 50 or 70%) sealed under nitrogen.
Storage: Stable for many months if stored at -20 C.
May be stored for shorter time at 4 C.
Characteristics of Pure Glutaraldehyde:
1. Clear. Yellow color indicates polymerization. Color should be no more than pale yellow.
2. pH above 3.5. Should be discarded if pH is below 3.5.
3. Spectrophotometric absorption at 280 nm. Other absorption peaks indicate impurities (usually absorb at 235 nm).
Preparation of Fixing Solutions:
Should be prepared and stored in clean, glass-stoppered bottles.
Stable for some weeks at 4 C in a refrigerator in the dark.
Freshly prepared final solutions should be used whenever possible.
Stock solutions:
Glutaraldehyde: Dilute glutaraldehyde to concentration twice that of desired final conc. (usually 2-6%) with ddH20.
Buffer: Prepare buffer solution with concentration twice that of desired final concentration (usually 0.05-0.2M). Adjust pH of buffer to desired pH. Adjust osmolarity of buffer to desired osmolarity with sucrose, glucose or NaCl.
Working fixative:
Mix 1 vol. of glutaraldehyde with 1 vol. buffer solution.
B. Formaldehyde
0
H-C
H
Advantages:
1. Rapid rate of penetration (more rapid than either glutaraldehyde or osmium tetroxide).
2. Reacts with proteins, unsaturated fatty acids and DNA. Very effective in fixing mucoproteins. Prevents extraction of glycogen if time of fixation is not prolonged.
Disadvantages:
1. Many reactions are reversible. For the most part fixative is removed with washing.
2. Only partially destroys osmotic properties of membranes. Osmolarity must be carefully adjusted when possible.
3. Specimens cannot be stored for any length of time without further processing.
4. Does not preserve soluble polysaccharides or acid mucopolysaccharides.
Preparation of Fixing Solutions:
Stock solutions:
Methanol-free formaldehyde prepared from paraformaldehyde-40% solution:
Dissolve 40 gm of paraformaldehyde in 100 ml ddH20 by heating to 65 C with continuous stirring.
Add a few drops of 40% NaOH until solution is clear.
Cool before using.
Store in clean glass-stoppered bottle in refrigerator in the dark. Stable for some weeks.
Buffer: Prepare buffer solution with concentration twice that of desired final concentration (usually 0.05-0.2M). Adjust pH and osmolarity of buffer to desired levels.
Working fixative:
To 50 ml of buffer add:
ml 40% paraformaldehyde Final conc.
2.5 1%
5 2%
7.5 3%
10 4%
Working fixative is stable at 4 C in the dark for some weeks. pH may drop slightly with time and should be tested before use.
C. Acrolein (Luft, 1959)
acrylic aldehyde 0
H2C=CH-C MW = 56.062
H
Advantages:
1. Penetrates and reacts faster than glutaraldehyde or osmium tetroxide.
2. Causes little shrinkage.
3. Reacts rapidly with proteins.
4. Good for fixation of large blocks of tissue.
Disadvantages:
1. Highly flammable and extremely reactive (explosive).
2. Highly toxic.
3. Strong tendency to polymerize on exposure to light, air and certain chemicals. Commercial preparations contain an oxidation inhibitor (usually 0.1% hydroquinone).
4. Solubilizes lipids.
Destroys most enzymatic activities.
Causes loss of some microtubules.
Preparation of Fixing Solution:
Preparations of acrolein should be stored in tightly stoppered brown bottles in cool place.
Acrolein fixatives are usually prepared with 10% acrolein in 0.025 or 0.05 M phosphate or cacodylate buffer.
D. Combination Fixatives. Combine advantages of 2 fixatives.
1. Glutaraldehyde-acrolein fixative:
1% acrolein
2.5% glutaraldehyde
0.1M buffer (usually cacodylate or phosphate) adjusted to desired pH and osmolarity
2. Glutaraldehyde-paraformaldehyde fixative: (Karnovsky, 1965)
2% paraformaldehyde
2.5% glutaraldehyde
0.1M buffer (usually cacodylate or phosphate) adjusted to desired pH and osmolarity
3. Glutaraldehyde-paraformaldehyde-acrolein fixative: (Hayat, 1970)
10% acrolein 3 ml
6% fresh paraformaldehyde 5 ml
10% glutaraldehyde 6 ml
0.2M buffer 5 ml
ddH20 1 ml
II. Permanganate Fixatives
Can be used as primary fixes or post-fixes after primary glutaraldehyde-acrolein fixation
A. Potassium permanganate (Luft, 1956) KMn04
Advantages:
1. Continuity of some membrane systems better preserved.
2. Preferred for some botanical studies (ex. seeds) due to ability to penetrate thick cell walls.
3. Some membranes seen with great clarity.
4. Glycogen is fixed and stained.
Disadvantages:
1. Extracts many cellular components including soluble cytoplasmic proteins, ribosomes, microfilaments, microtubules, and large amounts of lipids. Does not fix chromatin well.
2. Causes swelling of other cytoplasmic components.
Preparation of Fixing solution:
Stock solution:
1.2% KMnO4 in ddH20.
Store in well-filled, glass-stoppered bottle in refrigerator. Solution is slowly reduced on contact with air.
Working fixative:
Mix 1 part 1.2% KMnO4 and 1 part desired buffer adjusted to correct pH. 0.5% NaCl may be added to prevent swelling of cellular structures.
Fixative cannot be stored.
B. Sodium permanganate NaMn04
Essentially similar to KMnO4 but significantly better preservation of integrity and continuity of plasma membrane.
III. Osmium tetroxide
0 0 MW = 254.2
Os
0 0 Solubility in H20 = 7.24% at 25 C
Advantages:
1. Acts as electron stain as well as fixative.
2. Good preservation of cellular structures. Reacts with components, particularly lipids, that are not fixed by aldehydes.
Disadvantages:
1. Slow rate of penetration. Influenced by composition of buffer. Problem is reducead or overcome by stabilizing structures with an aldehyde primary fixative. Specimens must be very small.
2. Only partially preserves proteins. Most carbohydrates lost. Overcome by use of primary aldehyde fixative.
3. Soluble in certain lipids. Can be taken up in reduced form and subsequently reduced to form black deposits.
4. May cause clumping of DNA. Can be prevented by addition of Ca2+ or postfixation (en bloc staining) with uranyl acetate after OsO4.
5. Despite interaction with lipids, considerable extraction of lipids still occurs. Can be reduced by addition of 1-3mM CaCl2 or fixation at 0-4 C.
6. Destroys osmotic properties of membranes. Unpreserved cellular components free to diffuse out during prolonged fixation. Can cause swelling or shrinking: influenced by tonicity and type of ions present.
7. Very toxic and volatizes readily at room temperature.
Commercial availability:
Sealed vials of crystalline OsO4 (0.1 to 1 gm).
Aqueous 4% solutions
Storage:
Store in clean, well sealed glass containers.
Stable for some months if stored away from sunlight and organic matter. Strong oxidizing agent. Reduced in presence of organic matter or sunlight. Black color indicates reduction of 0s04.
A. Osmium tetroxide fixative
Preparation of Fixing Solutions:
Stock solutions:
Dissolve 0s04 in ddH20 to concentration at least twice that of desired final concentration (final concentration usually 2-4%). 0s04 dissolves slowly in H20. Can be accelerated if solution is agitated.
Buffer with concentration twice that of desired final concentration adjusted to desired pH. Concentration of 0s04 must be considered when adjusting osmolarity.
Working fixative:
Mix 1 part stock 0s04 solution with 1 part buffer solution.
B. Glutaraldehyde-osmium tetroxide fixative:
Combines advantages of both fixatives when added together. Prevents some extraction of components seen when glutaraldehyde fixative is followed by osmium tetroxide fixative.
Fixation solution is unstable. Cannot be stored since glutaraldehyde and osmium tetroxide react wtih each other.
Preferable to carry out fixation at 0-4 C to reduce reaction between glutaraldehyde and osmium tetroxide.
Working fixing solution:
0.8% glutaraldehyde
0.7% osmium tetroxide
0.1M cacodylate buffer
Another method that has been used is the brief fixation of cells in glutaraldehyde followed by the addition of a small amount of osmium tetroxide to the solution.
IV. Uranyl acetate
While used primarily as a stain, uranyl acetate has some fixation properties and may be used as a post-fix after glutaraldehyde and/or osmium tetroxide.
Particularly good as a fixative for membranes. Prevents clumping of DNA.
Disadvantages:
1. Glycogen extracted or rendered unstainable.
2. Cannot be used with phosphate or cacodylate buffer. Specimens must be washed thoroughly after use of these buffers before placing in uranyl acetate.
Working fixative:
0.25 to 2% uranyl acetate in ddH20 or maleate buffer. Final pH is in region of 5.
This procedure is also called en bloc staining.