Refractive Index & Diffraction
The refractive index (RI) of glass is about 1.52, although it may vary a little depending on the type and quality of the glass. The RI of glass coverslips would be much the same. For total transparency a mounting medium with a RI that is the same as the glass is necessary, then the differentiation between different tissue elements would be dependent on the staining alone. If the RI of the mounting medium is greater or less than the RI of the glass, optical diffraction takes place and the object being viewed ceases to be completely transparent. Tissue elements can be seen to a greater or lesser degree depending on the amount of diffraction present, but many membranes and other structures can be seen.
Diffraction is sometimes introduced deliberately. An example is the lowering of the substage condenser when examining a wet preparation of a urine sediment. Many cells, crystals and bacteria can be identified very easily in this manner. However, it is quite rudimentary and not always appropriate, cytoscreening of cells in urine for malignancy is an example, and procedures which allow for mounting with a high RI medium are necessary to permit a detailed examination.
Resinous vs. Aqueous Mounting Media
The RI quoted for mounting media usually refers to the fluid itself rather than of the mounted preparation. When first coverslipped, the medium may not have fully penetrated the tissue, but this is usually quickly remedied. As the mounting medium hardens, that is, as the solvent evaporates, there is often a slight rise in the RI of the preparation. At this point some mounting media may precipitate and obscure the tissue detail. That is why ringing is necessary, in which case the RI can be expected to remain the same for some time. In diagnostic settings ringing is not as popular as in the past but it is an effective way to preserve the integrity of the mount. Whether ringed or not, important preparations with significant findings should be photographed and stored archivally.
In general, resinous media dissolved in xylene or toluene have a RI very close to that of glass and produce preparations that are completely transparent. Watery mounting media usually have a lower RI than resinous media. The RI of gum and gum sugar solutions is usually in the high 1.4 something range, but varies depending on the precise ingredient mix being used. In general, the higher the concentration of the ingredients, the higher the RI. Glycerol, both absolute (RI 1.47) and diluted (lower), is the lowest. However, most of the media produce preparations with RIs not too far from that of glass and are well within the working range of a modern microscope.
The most appropriate way to select mounting media is to use them, then choose those that are found to be satisfactory for the purposes for which they are to be used.
Ringing is a procedure for sealing the edges of coverslips so that the mounting medium is no longer exposed to the air and evaporation of the solvent is inhibited. The ringing media harden fairly rapidly trapping the fluid mounting medium under the coverslip. The name comes from the way it was originally done, i.e. with circular coverslips and a turntable. The coverslipped preparation was centered over the turntable so that it slowly revolved with the edge of the coverslip rotating without any deviation from the center. The ringing medium was then applied with a fine brush in a ring around the coverslip. With square and rectangular coverslips more common today the medium is often applied with a straight metal rod rather than a brush. Often this is to make the preparation aesthetically pleasing.
Traditional ringing media are applied either as a varnish which dries by evaporation or as a solid which is melted for application, then sets hard as it cools. Once ringed, preparations are considered permanent. An often substituted alternative for occasional use is nail varnish. This is available in a wide variety of colours, including black, and comes with a brush for application. Drying time is a few minutes as the solvent evaporates, but more than one coat may need to be applied. Keep in mind that trace amounts of the solvent may diffuse into the mounting medium before the varnish dries, so check its suitability first.
Ringing media are better purchased than compounded.