Fixation of tissues is the most crucial step in the preparation of tissue for observation in the transmission electron microscope. Fixation consists of two steps: cessation of normal life functions in the tissue (killing) and stabilization of the structure of the tissue (preservation). The goal of fixation is to preserve structure as faithfully as possible compared to the living state.
The three most important parameters to remember about fixation are:
(1) keep the time between killing and fixation to a minimum.
(2) keep the size of the tissue as small as possible without losing information or destroying the tissue. If a large specimen must be fixed, keep one dimension less than 1 mm, or else nick areas of the specimen that can be discarded so that the fixative can penetrate. Effective penetration of fixative is about 0.5 mm for osmium.
(3) keep gross tissue deformation to a minimum by using sharp implements and keeping manipulation of the specimen to the minimum necessary.
Dehydration
Dehydration is the chemical removal of water from the specimen. Common dehydrating fluids are ethanol and acetone. The potential problems of dehydration are shrinkage of the specimen, plasmolysis, and removal of soluble components from the specimen. Dehydration must be conducted relatively rapidly in order to prevent excessive extraction of alcohol and acetone-soluble compounds, but slow enough to prevent plasmolysis.
Extraction of specimen components is difficult to control. Low molecular weight carbohydrates are particularly susceptible, since carbohydrates are usually poorly cross-linked if at all following fixation. Proteins tend to be cross-linked by glutaraldehyde during primary fixation and the lipids by osmium tetroxide during secondary fixation. The carbohydrates are essentially unfixed. Linked to the problem of extraction is that of shrinkage. Both problems are most serious at low concentrations in the dehydration series. In general, rapid dehydration is best for these reasons.
By 70% alcohol, the tissue no longer shrinks as much, but does begin to harden. In fact, extended periods of dehydration in the higher concentrations of alcohol may make the tissue quite brittle. If a stopping point is needed, most histologists choose 70% to 100% alcohol as a good place to stop for the evening.
If there is evidence of plasmolysis, perhaps additional dehydration steps (and/or longer changes) may be required. Cell membranes sometimes retain some osmotic activity after short periods of fixation. Longer periods of fixation in glutaraldehyde can reduce osmotic sensitivity as well. (Membranes are essentially insensitive to osmotic changes after 48 hours of fixation in glutaraldehyde.) Poor fixation will aggrevate problems with dehydration.
Dehydration at refrigerator temperatures slows the process down a bit and tends to lend some rigidity to the tissue. It may also reduce plasmolysis slightly. Plants are the most sensitive to poor dehydration, and therefore, refrigerated dehydration is preferred for these tissues.
When changing solutions, make sure that the specimen does not dry out. Therefore, most workers do not pipet the bottom of the vial dry between changes, but leave a little liquid to keep the specimens dry. On the basis of relative volumes, this remaining solution is inconsequential.
To mix alcohol solutions, 95% alcohol is used because using 100% would be prohibitively expensive. Using 95% alcohol to make the dehydrating fluids can be confusing even with the aid of a calculator. Therefore, histologists have developed a short-cut for these calculations: the amount of alcohol needed is calculated as if you were using 100% (for instance, to make about 100 ml of 70% alcohol, you add 70 ml to a graduated cylinder). Then the final volume of the solution is reduced by 5%. (In this example, 25 ml of water is added, making 95 ml of 70% alcohol rather than 100 ml). This is as accurate as adding the 73.68 ml of 95% alcohol which would be needed to make 100 ml of 70% solution, and it is much faster.
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