MERITS & DEMRITS OF

DIFFERENT FIXATIVES

FIXATION Fixation is a critical step in the preparation of histological

sections by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction.

FIXATIVES

These are different agent employed in the preparation of histologic or pathologic specimens for the purpose of maintaining the existing form and structure of all of the constituent elements.

A large variety of fixatives is now available .Each fixative has advantages and disadvantages, some are restrictive while others are multipurpose

Different Fixatives

SIMPLE FIXATIVES:1. FORMALIN:

ADVANTAGE:1. Rapid rate of penetration (more rapid than either glutaraldehyde or osmium tetroxide).2. There is no causes of shrinkage

DISADVANTAGE:1.Due to long storage specially in cold weather , white precipation is formed in the Formalin called PARAFOR MALDEHYDE 2. due to formation of PARAFOR MALDEHYDE , Formalin itself loose its concentration .

2. GLUTARALDEHYDES:

ADVANTAGE: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%. DISADVANTAGE: 1.Does not destroy osmotic properties of cells. Osmolarity of fixative must be carefully controlled..2.Can cause shrinkage of cells at higher concentrations.

CONTI…

4. Tends to polymerize at alkaline pH (above 7.5). 5. Has no staining action. 6. 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.

3. ACROLEIN: ADVANTAGE: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. 

DISADVANTAGE: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.5.Destroys most enzymatic activities.6.Causes loss of some microtubules.

4. POTASSIUM PERMANGANATE: ADVANTAGE: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. DISADVANTAGE: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.

5. OSMIUM TETROXIDE: ADVANTAGE: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.

DISADVANTAGE:1.      Slow rate of penetration. Influenced by composition of buffer.  Problem is reduced 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. 

CONTI…..4.   May cause clumping of DNA.  Can be prevented by addition of Ca2+ or post fixation (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.

6. URANYL ACETATE:

ADVANTAGE:1. 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.2. Particularly good as a fixative for membranes.  3. Prevents clumping of DNA.

DISADVANTAGE: 1.  Glycogen extracted or rendered unstainable. 2.  Cannot be used with phosphate .  Specimens must be washed thoroughly after use of these buffers before placing in uranyl acetate.

 

COMPOUND FIXATIVES:7. Zenker’s Fluid: ADVANTAGE:1.It preserves nuclear and connective tissue very well .2.Fixation in Zenker’s fluid facilitates metachromatic staining . 3.Fixation time is 10-20 hours . DISADVANTAGE:1.Only thin pieces of tissue can be fixed ( can penetrate up-to 5 mm) .2. It is expensive and has to be preserved only in special bottles made of nickel alloy .3. It deteriorates fast . 4.If the tissue is left in the fluid for 3-4 days , it becomes hard and brittle .

8. Bouin’s Fluid: ADVANTAGE:1.Rapid penetration , preserves glycogen , improves greatly staining of nuclei and connective tissue . 2.The tissue takes-up yellow color of picric acid , hence small piece of tissue can easily be spotted

DISADVANTAGE:1.It over hardness the tissue if left in the fixative for more than 24-hours .2. It lyses red cells . 3. Tissue fixed in Bouin’s fluid should be directly transferred to 70% alcohol . 4.It must not be washed in water because it forms water-soluble picrate .

.