Bhaskar Ganguly Ph.D. Scholar (Vety. Biochemistry) Animal Biotechnology Center Deptt. Of Vety. Physiol. & Biochem. C.V.A.Sc.

Pantnagar. INDIA

FIXATION
Fixation is an attempt at stabilizing biological systems with minimal distortion to their cytomorphology and cytochemistry. Biological electron microscopy employs two methods for obtaining these results, chemical and/or physical fixation. While chemical fixation remains the most common method of specimen preservation, physical techniques are gaining in popularity. Procedures combining chemical and physical fixation are, in certain procedures, both useful and advisable.

Chemical Fixation
It prepares the cell for a whole series of rather

traumatic events imposed by the physical characteristics of the electron microscope, (i.e. improved resolution, extreme vacuums and the intense heat of the electron beam). Ideally, the reagent selected as a fixative should transform the viscous colloidal protein solution (protoplasm) into stabilized elastic gelatin. The spatial relationship of all organelles should be maintained; the phospholipids, which form the framework of the cell, should be stabilized and the remaining chemical constituents rendered insoluble.

Chemical Fixation (contd)

Some of the properties that should be taken while selecting or preparing a fixative are: Rate of penetration - Determines the speed at which the reagent affects the cells structure and function. The rapidity of this process is paramount to good fixation. Low molecular weight fixatives that penetrate the cell rapidly are often recommended. Buffers - Used to maintain the desired pH. Most cells are accommodated very well in the range of 7.2 - 7.4. Certain highly hydrated tissues prefer a more alkaline pH (i.e. 8.0 - 8.4), while nuclear material and the fibrils of mitotic spindles appear to favor the more acid pH (i.e. 6.0 - 6.8).

Chemical Fixation (contd)

Tonicity - Hypertonic solutions cause shrinkage while hypotonic

solutions induce swelling. Tonicity of a solution can be adjusted by electrolytes (e.g. sodium chloride) or non-electrolytes (e.g. sucrose). Electrolytes are favored because non-electrolytes tend to decrease the rate of penetration and increase cellular extraction. Most fixatives are preferentially prepared slightly hypertonic. Temperature - Fixation is usually initiated at a low temperature (e.g. 4 C) and allowed to slowly rise to room temperature. Reduced temperatures diminish leaching of cellular components and aid in preserving some enzymatic activity. Lowered temperatures inhibit enzymatic activity, thus preventing catabolism. Size of sample - Determines the quality of fixation. In general, the size of the sample should not exceed 0.5 mm3 to assure good penetration and fixation.

Physical Fixation
Physical fixation utilizes extremely low (-196 C.)

temperatures, applied to very small samples, to provide a frozen slice of life. Freeze drying and/or freeze etching are two physical techniques commonly used to circumvent some of the adverse effects inherent in chemical fixation. The major attributes of these techniques are the rapidity with which they fix the sample and often their failure to kill the cells. Freezeetch replicas have, in particular, been especially useful for producing three dimensional images and the new information gained on membrane fine structure.

Chemical - Physical Fixation

It is sometimes advisable to combine chemical

and physical fixation for specific samples. A rare sample may be lost forever if the nonchemically treated cell is inadvertently lost or damaged during physical processing. Others may require additional support to withstand collapse in the high vacuum of the instruments employed. Finally, as physical fixation does not always kill a cell this phenomena assumes major importance in laboratories using live pathogens.

Fixation (contd)
4 different methods of fixation are: 1.Vascular Perfusion - gives best results; fixative is perfused in live animal replacing blood from the circulation 2.Immersion Fixation - easiest; tissue is separated from the animal and dipped in the fixative 3.Dripping Method - the live animal is anesthetized and dissected to expose the tissue, which is then flooded with the fixative 4.Injection Method - the organ or tissue of interest is exposed and injected with the fixative; the organ is cut and removed, cut to smaller pieces and further fixed

DEHYDRATION
Chemically fixed tissue should be thoroughly washed with a buffer solution before introducing a post-fixative or a dehydrating agent. This step minimizes the possibility of a reaction between various chemical reagents. It is also recommended that sucrose be added to the buffer to maintain the same osmolarity as the fixative.

Organic Dehydration
It is the complete removal of all water with a solvent miscible

with the final embedding media. Ethanol and acetone are the two common organic agents. Shrinkage is minimized by using dehydrant in increasing concentrations. Cooling the dehydrant can reduce some cellular extraction, but care must be taken to eliminate possible water uptake via condensation. The time allotted should allow good exchange of the solutions but disallow extraction. Some embedding media are immiscible or only partially miscible in either alcohol or acetone. This necessitates the introduction of an intermediary transitional solvent which is miscible with both the dehydrating agent and embedding media. The two most frequently used transitional solvents are styrene with acetone or propylene oxide with alcohols.

Dehydration with Watermiscible resins

This technique employs a water miscible resin,

usually Durcupan or glycol methacrylate, as the dehydrating agent and a compatible water immiscible resin as the embedding media. While the major advantage claimed in this technique is the by pass of the organic solvent in dehydration, it should be noted that many of the monomers used in final embedment are also organic solvents. The techniques major advantage appears to be in the area of histochemistry and cytochemistry.

EMBEDDING
Embedding includes the removal of the dehydrating agent or transitional solvent and the infiltration of the cells interstices by a resin monomer. In selecting a resin, consideration should be given to numbers of qualities which are deemed essential for a good embedment. The process comprises of infiltration of the embedding agent and its successive polymerization.

Embedding (contd)
Some of the properties of a good embedding agent are: Low viscosity monomer for rapid and homogeneous infiltration Chemically inert with respect to the cells Miscible with the dehydrating agent or the transitional solvent Should produce uniform polymerization with the accelerator or catalyst without causing translocation of cellular components The embedment should section easily Should not interfere with the heavy metal stains used to increase the contrast Should be highly electron transparent and stable under the beam

Embedding (contd)
Rate of Infiltration depends on: Viscosity of the infiltrating medium Nature of previous agents used (fixation and dehydration, accumulation of heavy metals) Temperature & Humidity Mechanical agitation Calcification of tissue; presence of cavities/ air bubbles (e.g. in bones) Polymerization of embedding agent is achieved by Heat or Ultraviolet irradiation. Polymerization can be facilitated by the presence of Curing Agents, viz. cross-linking agents (hardeners) and catalysts (accelerators). Hardeners (e.g. organic anhydrides) form cross-linkages between the resin monomers. Catalysts (e.g. tertiary amine compounds) promote epoxy-epoxy, epoxy-hydroxyl and epoxy-curing agent linkages.

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