Different types of mounting medium for microscopy

Mounting media are needed for making permanent slides. The mounting medium holds the specimens in place between the cover slip and the slide. The choice of the right mounting medium is a separate topic all on its own. There are countless commercial and home-made mounting media available. Which ones should one use? In many cases the microscopist has no choice: some specimens simply require the use of a specific mounting medium, otherwise the structure that one wants to observe is not properly visible. Alternatively, not all specimen types are chemically compatible with the solvents of the medium.

Generally, mounting media for permanent slides can be categorized into water-based and organic solvent based mounting media. While many water-based mounting media for permanent slides solidify and hold the specimen firmly in place, some others remain in a liquid state. In this latter case, it is necessary to prevent the liquid from flowing out by sealing the four sides of the cover slip. Nail polish can be used for this. In the following paragraphs, I’d like to give you an overview of the different types of mounting media.

Water-insoluble mounting media that solidify

Euparal: This mounting medium was invented in 1904 by Prof. G. Gilson, Professor of Zoology at Louvain University, Belgium. It contains the substances sandarac, eucalyptol, paraldehyde, camphor, and phenyl salicylate. Euparal possesses a nice odor (but don’t smell it anyway), due to the natural oils that are included. Euparal is commonly used to mount histological specimens and insects. One big advantage of Euparal is, that the specimens can be transferred directly from the alcohol in which they are stored. Do not embed specimens which contain water, this may result in a clouding of the mounting medium.

Summary: Advantages of Euparal include the possibility to directly transfer specimens from alcohol to Euparal without the need of toxic solvents. A disadvantage is the relatively long drying time of a few days to weeks.

Canada Balsam: This is a natural mounting medium obtained from the balsam fir tree (Abies balsamea). The optical properties are nearly identical with those of glass. For this reason, Canada Balsam was used for many years as a kit to hold optical lenses in place. Meanwhile, synthetic lens kits have replaced Canada Balsam, it is still used as a mounting medium for microscopy, however. Canada Balsam has the advantage that its optical properties do not deteriorate with age. Permanent slides mounted with Canada Balsam have been stored for a century and are still useful.

The disadvantage of Canada balsam is, that the specimen must be placed into xylene (toxic!) before embedding. Wet specimens must first be dehydrated in alcohol and then transferred to xylene. Transferring specimens directly from alcohol to Canada balsam won’t work, because the alcohol won’t dissolve the Canada balsam.

Summary: The advantage of Canada balsam is the long storage ability of the slides. Other, modern, mounting media may have a similar storage ability, but with Canada balsam there is historic experience. A disadvantage is the need for toxic solvents when preparing the specimen. Apparently, it is also not very cheap to obtain.

Eukitt and other resin-based media: Eukitt is a very fast drying general-purpose resin-based mounting medium. Eukitt will solidify within about 20 minutes. The specimens must be free of water and placed first in alcohol and then in xylene prior to mounting. The use of xylene is a disadvantage, as it is harmful when inhaled. Eukitt itself can also be diluted by xylene to adjust it viscosity.

Besides Eukitt, a range of other resin-based mounting media are commercially available, such as Diatex, Entellan, Malinol, Rhenohistol and Depex. They differ in their refractive index. All of these mounting media require the specimen to be first dehydrated in alcohol and then transferred to xylene. Some of these resins shrink significantly during the drying process.

Summary: The advantage of Eukitt is that it is a fast drying mounting medium. The disadvantage is the need for toxic solvents to prepare the specimen.

Clear nail polish: Nail polish can be used to seal the sides of the coverslip when using aqueous mounting media. It can also be used directly as a mounting medium. The specimens must first be dehydrated in alcohol and can then be directly mounted (without xylene) in nail polish.

Summary: The advantage of nail polish is, that it is readily available and that it avoids the use of toxic organic solvents to treat the specimens. One disadvantage is, that it seems to shrink a lot when making very thick mounts (such as whole insects).

Water-insoluble mounting media that remain liquid

While it is possible to use various oils (immersion oil and paraffin oil) as a mounting medium, they are generally not used to make permanent slides. The specimen must be dehydrated with alcohol and then transferred to xylene so that the liquid mounting medium (the oil) is able to reach all the parts of the specimen. I can imagine that it is this xylene which causes a problem with the sealing of the cover slip, by preventing hardening of the nail polish used for sealing.

Water-soluble mounting media that solidify

Glycerol jelly: This is a water-based (aqueous) mounting medium. There are several variations to the recipe, fine tuned for specific mounting applications. The classical recipe according to Kaiser (1880) includes Phenol as an antiseptic, so it hazardous for the use in schools and at home. The handling of this mounting medium, is also not too easy. The bottle with the solid glycerol jelly must first be warmed in a water bath to make it liquid. Do not make it too hot, otherwise it will not solidify any more. The specimen is submerged in the warm jelly and the cover glass is placed on top. Bubbles are a problem with this medium. The edges of the cover glass now must be sealed with nail polish to prevent drying out.

Glycerol jelly is one of the most difficult mounting mediums to use, but sometimes there is no other satisfactory alternative to an aqueous mounting medium. Water-based mounting media are useful for making permanent mounts of water organisms, algae, protozoa, etc. Glycerol jelly according to Kisser (not Kaiser) is commonly used to preserve pollen samples. Treating some specimens with organic solvent-based mounting media would cause them to shrink or change their shape in other unacceptable ways. Solvent-base media may also dissolve some of the pigments, such as chlorophyll, from the specimen, which does not happen when using aqueous media such as glycerol jelly.

Summary: The advantage of Glycerol jelly is that it s water-based and that this avoids the need of alcohol dehydration (which possibly deforms the specimens), and other toxic organic solvents. Some specimens can only be satisfactorily mounted in Glycerol jelly. It also does not shrink. The disadvantages include the need for a potentially toxic antiseptic in the jelly, the difficulty of mounting the specimens and the need to seal the cover slip with nail polish.

Water-soluble mounting media that remain liquid

Glycerol: It is possible to make a permanent mounts by embedding the specimen either in pure liquid glycerol or a specified glycerol-water mixture. The glycerol-water mixture can be adjusted to an appropriate refractive index. Adding more water lowers the refractive index. It is also possible to use pure water alone (for some delicate algae, for example).

Algae and other water organisms can be embedded this way. Algae that are embedded in pure glycerol may shrink because the glycerol withdraws water from the cells. If the algae shrink too much, then the glycerol should be more diluted with water. A high concentration of glycerol should be maintained, however, otherwise there is a risk of fungal growth in the medium.

Making liquid permanent slides is somewhat more advanced. The drop of glycerol must be very small so that it will not touch the sides of the cover slip. On all sides, there should be a few mm of air between the sides of the cover slip and the glycerol. The sides of the cover slip are then sealed with nail polish two or three times to prevent glycerol from leaking out. Here it is very important that the glass surfaces are completely clean and have not been in contact with glycerol, otherwise the nail polish will not hold.

Summary: The advantage of glycerol is, that fungi and algae do not shrink as much as with other mounting media. It is also not necessary to treat the specimens with alcohol or organic solvents, which may introduce artifacts and remove pigments. The disadvantage is, that it is difficult to prepare slides that are truly permanent in nature. A proper sealing of the cover slip corners is absolutely necessary if one wants to store the slides over extended periods.