Ranunculus pollen mounted in air, no cover glass.

Ranunculus pollen mounted in air with cover glass.

Ranunculus pollen mounted in water.

Ranunculus pollen mounted in Euparal. The pollen grains started to shrink.

Ranunculus pollen mounted in clear nail polish. The pollen grains show signs of significant shrinkage.

The mounting medium can have a significant effect both on the image quality and on the specimen itself. I tried a little experiment by observing pollen from a plant (in this case the buttercup, Ranunculus), mounted in five different ways:

• Air-mounted, with no cover glass
• Air-mounted, with a cover glass
• Mounted in water (temporary mount)
• Mounted in Euparal medium (permanent mount)
• Mounted in nail polish (permanent mount)

All observations were made using a 20x achromatic objective.

Results

The images on the right show that the mounting method has a significant impact on the way that the pollen grains appeared. The results can be summarized as follows:

• Air-mounted specimens show the least details. The pollen grains show a thick dark fringe, which covers much of the details. This is due to the large difference in refractive index between the pollen grains and the surrounding air. Opening the condenser diaphragm reduces the dark fringes, but also lowers contrast and depth of field. The cover glass presses the pollen against the slide, so that more of them are in focus. Otherwise the cover glass did not seem to make much difference.
• The water-mounted sample provides a much better image. The dark fringes are now gone, due to the similar refractive index of the pollen and the medium. The pollen appear spherical, because the water causes them to swell up.
• Pollen mounted in Euparal started to shrink and therefore appear smaller in size. Kinks and folds are also visible. These artifacts are produced because the (non-water based) Euparal has withdrawn moisture from the pollen.
• Clear nail polish showed a similar, but more pronounced effect as Euparal. The deformations of the pollen are very clearly visible. Evidently the solvent of the nail polish also removed significant amounts of water from the specimen. The nail polish itself lost some of its volume during drying and started to shrink as well. Air bubbles also became visible in the nail polish. Irregular drying of the mounting medium and a change in the shape of the mounting medium during drying can lead to shear-forces, which may distort the shape of the specimen.

What about Glycerin Gelatin (glycerol gelatin, jelly)?

Glycerin Gelatin is a water-based mounting medium. Glycerin Gelatin according to Kisser is one of several Glycerin Gelatin variations. It is a common medium for mounting pollen. Due to its water-based nature it does not cause the pollen to shrink. I’ll add a picture of this, when I have some of this mounting medium available. An alternative water-based mounting medium is fructose syrup. Both Glycerin Jelly and fructose syrup do not dry completely and therefore require a sealing of the sides of the cover slip with nail polish (but the pollen do not touch the nail polish).

Lessons learned

What can we learn from these observations?

• First, permanently mounting a specimen is not only important for slide storage. The mounting medium significantly influences the transparency, resolution and shape of the specimen.
• Second, the choice of the mounting medium depends on the type of specimen to be observed and on the type of microscopic technique to be used. For phase-contrast work the refractive index of the mounting medium should be different from the refractive index of the specimen. For bright-field work the refractive indexes should be similar. Large differences in refractive index can lead to the dark fringes as seen in the air-mounted specimens.

Some philosophy

So which mounting medium now results in pollen grains with a “true” or “correct” shape? The problem now is: what is the “correct” shape? Biological specimens may change their appearance depending on the environment. After a rain shower, the pollen may have a more roundish appearance, after having osmotically absorbed much liquid. Pollen that has dried in the air may resemble more the shape of the Euparal and nail polish samples. The choice of the mounting medium may therefore even include these considerations.

External Links, References

• An introduction to pollen analysis
• Aqueous Mounting Medium Protocols
• Making and Using Aqueous Mounting Media

Ranunculus repens pollen in dark field

Ranunculus repens pollen in bright field

Ranunculus repens

Now a few words concerning sample preparation. The pollen was collected by dusting the flower over a microscopic glass slide. The pollen was briefly dried in open air (about 1 hour) and then permanently mounted in Euparal mounting medium. The standard mounting medium for pollen is Glycerin gelatin, which is water based. I assume that the drying and the Euparal caused the pollen to shrink somewhat, but I yet have to make a comparison with fresh pollen.