Stacked image of dandelion pollen in dark field

The pollen grains from a dandelion (Taraxacum sp.) were collected and air-mounted (no liquid mounting medium used). Eleven separate images were stacked together to increase the depth of field and to produce the final image. The color contrast was then adjusted. Dark-field patch stop was used.

Yeast

You can either use fresh (wet) yeast or dried yeast. In either case, take a small amount and dissolve in a little bit of water, until the liquid becomes turbid. Use this suspension for microscopy. (read The hemocytometer (counting chamber) to sell how yeast cells look like in a counting chamber).

Yogurt

One of the more difficult specimens. Yogurt contains many bacteria, these are a bit difficult to see with bright-field microscopy. You can stain them (read ). Take a small sample (knife-tip) and dissolve in water. Then apply a drop to the slide, apply a cover glass, and observe under the microscope.

Cheese

Here you have to take the right kind of cheese. The toast-cheese (the one where each one is wrapped separately in plastic foil) won’t work. They don’t have any fungus growing on them (Do not let it rot, you may be growing poisonous fungi). I’m a cheese lover and I consider Camembert, Brie, Gorgonzola blue cheese not only good for eating but also a valuable source for the fungi Penicilium.

Honey

Some of them contain pollen. If the honey is turbid (opaque) then this may be due to sugar crystals or due to pollen. Clear honey won’t work.

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.

There are several methods of preparing pollen grains, each one offers advantages and disadvantages. I can not give a general rule, it simply depends on the goal of the investigation and on the sample investigated. Pollen from wind-pollinated plants taken from a dry environment are probably best left in a dry condition, and not mixed with a water-based mountant, which may cause them to swell (depends on the osmotic potential of the medium, however). On the other hand, the obtained image quality and resolution may not be satisfactory in such a dry mount. It is a compromise, in which several factors have to be taken into consideration. A microscopy enthusiast, who does not need the slides for identification purposes, will again set different standards (such as avoidance of toxic solvents). People who want to publish their results, in turn, may have to rely on the preparatory technique which is customary in their field of research, for reasons of comparison. I recommend that the different methods are tried out.

Mounting techniques

Glycerol wet mount: Place a small drop of glycerol on a clean slide and tap the anthers of the plant so that the pollen falls into the glycerol. If necessary, carefully separate large chunks of pollen grains by stirring. Place a cover slip on top and seal the sides of the cover slip with nail polish. Use a very small amount of glycerol to make sure that the nail polish has enough area to stick the coverslip to the slide. Glycerol wet mounted slides can be stored for months if there is no leakage. The glycerol will withdraw water from the pollen. If the pollen is not dry, then there is a possibility of the pollen to shrink.

Air mounts (dry mounts): In this case, no liquid mounting medium is used. A cover slip is placed on top of the pollen grains and sealed on the side, either with nail polish or with tape. Nail polish may flow very quickly between cover slip and slide, so it may be best to use a nail polish of low viscosity (by letting some solvent evaporate first).

Glycerol jelly (according to Kisser): This is a very popular mounting medium for pollen. It is phenol-free (antiseptic additive) and therefore non-hazardous. It contains 10g of gelatin, 35ml distilled water and 30ml of glycerol (glycerin). After mounting, the sides of the cover slip need to be sealed. Due to the lack of an antiseptic, it is also necessary to work in a sterile manner, otherwise there is the risk of fungal growth in the medium. Maybe it is a good idea to treat the pollen grains first in alcohol to reduce the chance of fungal contamination by spores. Alternatively, one could experiment by increasing the concentration of glycerol.

Non-water-based mounting media: Euparal is a mounting medium which is not water based. Specimens which are present in alcohol can be directly transferred to Euparal. Place a pollen suspension on the slide and let the alcohol evaporate. Before mounting pollen in Euparal, I recommend that the pollen are first washed in alcohol and then compared to the original shape. Does washing in alcohol result in an unacceptable shrinking of the pollen or unacceptable loss of pigments? If not, then mounting the pollen in Euparal may be an alternative.

Reading materal

I found the following article: Marvels of pollen shown by your microscope (Popular Science, September 1939)
(The article recommends the use of organic solvents (such as xylol/xylene and others) to remove oil from the pollen. I do not recommend this due to health reasons, especially when preparing samples for educational purposes. Still, it gives a nice overview of the topic.)