To produce prepared microscopical slides of good quality often the refractive index of the mounting medium is often required. In case of stained sections the refractive index should coincide with that of the stained material, that means, the unstained material should be invisible in the mounting medium to suppress the blurring "refraction image". On the other hand, in case of diatoms the refractive index should be significantly higher than 1.45, which is the refraction index of the frustules. The difference should be 0.3 units at least. When investigating cruder forms like radiolarians or foraminifers, a smaller difference is desired to increase transparency. Unfortunately, Abbé refractometers are outrageously expensive, but the much simpler methods will also do..
Fix a vertical stick of 30 cm on a plate of wood of about 20x20 cm and cover the plate afterwards with a sheet of graph paper. Then fix a cavity slide horizontally tothe stick. Cover one quarter of the cavity with a coverslip and fix it in this position with a hot mounting medium (Fig.1). Now the cavity together with the coverslide forms a small hollow prism. Later, one drop of the liquid to be investigated will be put into this prism.
Measuring procedure
Fill the cavity with the liquid under discussion and move your eye in such a way that the zero-mark on the graph paper is seen at the very rim of the cavity, but just outside (blue line). Then move along the graduated line with the tip of a pencil and stop when the tip just appears inside the rim (red line). Note the position the tip of the pencil has touched. Using a calibration curve the value so obtained defines the refractive index. If the index of the medium is smaller than that of glass the two lines displayed in the graph are swapped. In this case the value obtained must be counted negative. If both indices are identical, both lines coincide. Of course, first a calibration curve must be established. This is done by using liquids of a known refractive index. Usually this calibration curve is a straight line.
| Medium | nD |
| Water | 1,333 |
| Ethanol | 1,361 |
| Isopropanol | 1,378 |
| Chloroform | 1,449 |
| Toluene | 1,496 |
| Methylbenzoate | 1,517 |
| Clove oil | 1,544 |
| a-Bromonaphthalene | 1,656 |
| Methyleniodide | 1,744 |
This method requires a microscope with a mechanical stage and a condenser with a high aperture (three-lens system) which can be adjusted vertically with a gear drive, an eyepiece with a reticule (graduated scale), an object slide prepared in the way explained above and a special filter: Fix one halve of a razor blade to a filter glass in such a way that the cutting edge corresponds exactly with the diameter and put this filter into the filter support of the collector with the fixed blade in an upside position. Use a clear green filterglass or a yellow filterglass in combination with a sodium vapour lamp, if available, to suppress colour fringing.
Measuring procedure
The measurement is done with a 20x objective, if possible with a 40x objective or a 60x objective. In theory high magnification is preferable, but often blurring through colour fringing decreases accuracy. So the optimal magnification must be empirically tested.
Adjust the objectslide in such a way that the optical axis of the system passes the cavity from outside, focus on the upper surface of the slide, open the condenser diaphragm and move the condenser vertically, so that the sharp edge of the blade is distinctly in focus. Remember the position of the edge on the reticule of the eyepiece. Move the object slide until the optical axis of the system just passes the rim of the cavity from inside. The position of the edge will then switch to the left (negative difference) or to the right (positive difference, fig.2). Remember the new position. The difference of the two positions thus obtained presents the refractive index. Of course first a calibration curve must be established.
You need a cell as shown in fig.3; the distance of the inner surfaces should not exceed 0.5 mm. Treat the surface of the objectslide gently as well as the surface of the coverslide with an abrasive, then rub some black ink into the scratches thus obtained and clean the surfaces afterwards. These prepared surfaces must face one another. The coverslide must be fixed with two clamps, otherwise it will float, if liquids of high density are inspected. Fixing the slide with a mounting medium is not a good idea because in this case it is difficult then to clean the cell.
Measuring procedure
You need a microscope with a graduated micrometer drive. Make sure that this drive is linear and independent of the starting position of the stage! When measuring the rotation of the micrometer drive, keep in mind that the zero-position may be passed!
The method itself is simple: Determine
the virtual distance between the two surfaces by focussing on the black scratches.
Using a calibration curve you will easily find out the refrative index.