Physical and Chemical Mechanisms of Preparation Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 106

above-mentioned chemical reactions, causing acidification of the fixative mixture.
A buffer system is used to trap these H + ions. This buffer system is composed of
ionized substances that contain two salts, one salt and one acid, or one salt and one
base. The buffer system chosen must meet certain criteria:

- Efficacy in the selected area, depending on pH
- Good water solubility
- Good penetration in biological systems
- Reduced side effects. In particular, the toxic effects on cells must be prevented,
as well as precipitations due to the presence of some cations (in particular with
Ca 2+ ). The ionic equilibrium of the fixative in relation to the ionic balance of the
cell must also be monitored

The most commonly used buffers are mineral buffers (phosphates or cacodylate)
or organic buffers (s-collidine, P.I.P.E.S).
The pH level is chosen depending on the milieu intérieur of the tissue (e.g., blood
plasma). It is usually near neutral (7.2-7.4).

6.1.2 Molar Concentration

Differences in molar concentrations between the fixative mixture and the tissue
lead to movements of water and ions across the plasma membrane, which is
semi-permeable. The molar concentration of the fixative mixture is, as with pH,
a replication of that of the milieu intérieur of the cells and tissues. For mammals, it
is around 300 mOsm. The osmolarity correction is calculated for the entire fixative
mixture. It has a component due to the fixative itself, a component due to the buffer
system, and possibly an additional component (salt or sugar).
The semi-permeability of the plasmic membrane is not destroyed by the alde-
hydic fixatives, so the osmotic balance between the cell and the fixative must be
watched until osmic fixation. It is necessary to rebalance the rinsing vector liquid
after aldehydic fixation and compensate for the osmolarity of the fixative, which is
no longer in the mixture.

6.1.3 Ionic Concentration

There are many movements of ions within the cells; they influence the electrical
conduction between cells, muscular contractions, the entry and exit of molecules
through the plasmic membrane, secretion, etc. Therefore, it is important to create a
minimum of disturbances at this level. But the reference level is difficult to know,
since natural ionic movements are extremely rapid. In the absence of precise data, it
is necessary to be sure not to add too much of a single ion; therefore, mineral buffers
containing both sodium ions and potassium ions, or better still, organic buffers,
which are neutral from this standpoint, are used.
Furthermore, experience shows that the presence of divalent Ca 2+ or Mg 2+

cations in the vector liquid provides better membrane preservation.