Characteristics of a chemical fixative

A good fixing agent should fulfill several criteria:

• It must kill the specimen quickly: But be careful, some chemical fixing agents are toxic and are also harmful to the health of a person.
• It must preserve the structures of the specimen, without introducing deformations or other artifacts. Insects may pull together their appendages, making them more difficult to see. The structures should then be sufficiently stable to withstand the dehydration and mounting.
• It must enter the specimen well to react with all parts: This can be problematic with some specimens. Make sure that the specimen is sufficiently small. Alternatively it is possible to puncture the specimen (insects) so that the fixing agent can enter more easily. Some specimens may contain air bubbles which prevent the fixing agent to reach all parts. In this case it may be necessary to apply a vacuum to remove the air.

Types of fixing agents

Chemical fixing agents can be categorized into the following 4 groups:

• Alcohol and acetic acid: This combination denatures proteins. The alcohol also removes some lipids. This is probably the preferred fixing agent for hobbyists, because it is less toxic than some other fixatives.
• Aldehydes (such as formaldehyde – toxic!): these react with amino groups in the specimen.
• Oxidation agents: these react with lipids.
• Tanning agents: react with proteins and with amino groups.

The choice of the fixing agent must be carefully matched with the specimen. Some fixing agents (eg. alcohol) may result in the shrinking of the specimen and therefore introduces artifacts. Sometimes it may be necessary to gradually increase the concentration of the fixing agent in order to prevent the formation of artifacts, but this depends much on the type of specimen used. I can not give general advice here, and recommend that one consults specific laboratory manuals.

Using alcohol

For the hobbyist who wants to prepare a slide every now and then, keeping a whole set of different chemical fixatives is probably an overkill (and not healthy either). I keep a small bottle of 96% rubbing alcohol on my shelf, into which I drop the specimens, usually small insects, as they arrive. They will store nearly indefinitely in this solution. When For making permanent slides, I directly transfer them into Euparal mounting medium.

Pure alcohol (ethanol) is also suitable for fixing and storing plant specimens, without cell contents. The alcohol has the tendency to shrink the cytoplasm, but does not affect the cell walls. The alcohol also hardens the plant material, making it easier to cut with a microtome (which often removes the cell contents anyway).

Alcohol/acetic acid solution

Acetic acid (acetate) compensates the shrinking effect of the alcohol. The Carnoy Clarke solution uses 3 parts 92% rubbing alcohol mixed with one part pure acetic acid. The correct alcohol:acetate ratio should be fine-tuned experimentally. If the cytoplasm still shrinks too much, the recipe according to Farmer may be tried out (2:1 alcohol:acetate ratio). Fixing should take place for about 24 hours.

After fixing

There are two more steps necessary: the fixing agent has to be removed (washing) and the specimen has to be dehydrated. Several fixing agents are water-based and this water has be be removed before mounting them in a non-water based mounting medium. Dehydration is not necessary when mounting in a water-based mounting medium such as glycerin gelatin. Dehydration is commonly done by placing the specimen in successively higher concentrations of ethanol. Afterwards the specimen is transferred into a solvent which is compatible to the mounting medium. Some mounting media require the specimen to be submerged in xylene (toxic). Other mounting media are able to directly accept the specimen from the alcohol (Euparal). If one sees a clouding of the slide, then this can be an indication that there was still some water in the specimen.

Heat-fixing of bacteria

Bacteria are treated differently. They must not only be killed, but also physically fixed to the glass slide. Otherwise they will be washed off during the staining process. This method also works with cells collected from the inside of the cheek and water samples.

• Place a bacterial suspension on the slide and let dry. Dry gently, dry completely but do not heat, otherwise the cells may pop open.
• Pull the glass slide through the flame of a Bunsen burner (1-2 times). The specimen should not come into contact with the flame (specimen on top, flame on the bottom). This step is called “heat fixing”. It kills of the bacteria and binds them to the glass slide much like an egg to a frying pan. The glass slide should be so hot that you are just able to hold it in the palm of your hands without causing burns. Heat the slide too much and you end up burning the bacteria 8and destroying their structure).
• The bacteria can now be stained. Place a drop of the staining solution on the cold slide. Rinse off with water and dry it in air. Do not dry-wipe, you will remove the fixed bacteria. You can then observe the bacteria directly in oil immersion even without a cover glass. Place the immersion oil directly on the fixed and stained bacteria.

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

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.)

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.

Canada Balsam: This is a natural mounting medium obtained from the e 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.

Toxicity: Solvent-based mounting media (such as Eukitt and Canada Balsam) require the specimen to be in xylene prior to embedding. This substance is toxic. Other mounting media, such as Glycerol jelly, may contain hazardous antiseptics. This aspect of toxicity is something to consider when making permanent mounts either as a hobby or for educational purposes in schools. One should ask oneself, if one should not use other alternatives.

Refractive index: The correct refractive index (RI) of the mounting medium can be critical for seeing details of the structure. If one uses phase contrast microscopy, then the RI of the mounting medium should be very different from the RI of the specimen. For regular bright-field work with pigmented specimens, the RI should be the same. In an ideal world, the mounting medium should be matched with the type of specimen. For amateur or educational work, this may be of less relevancy, however. Some high-end microscope objectives are calibrated to be used for a specific RI of the mounting medium, otherwise the resolution is reduced.

Compatibility with specimen: Specimes which are kept in water should be transferred into a water-based mounting medium. Transferring them into a solvent-based mounting medium may result in a clouding of the resin. Likewise, specimens which are kept in alcohol should be transferred to xylene and then embedded in a solvent-containing mounting medium. Euparal allows the specimen to be present in alcohol.

Pigment stability: Some mounting media cause a fading of pigments and stains over time. If pigment stability is of relevancy, then one should use mounting media which do not react with the pigments of the specimen. In some cases a fading of pigments is desirable, however. This brightens the specimen and makes it more easy to observe.

Shrinkage: Some mounting media shrink when they dry. The effect is particularly noticeable when thick specimens (e.g. whole insects) are embedded. Non-water based mounting media are known to do this. Glycerol jelly, which is water-based, does not shrink, however.

Durability: How long should the permanent slides be stored? Non-solidifying mounting media may not hold the specimen in place very well and there is the risk of running out if not sealed properly. Other mounting media may start to crystallize over the years. Still others may adversely react with the pigments of the specimens. Canada balsam is known for its good durability.

Cost: Some mounting media (such as Canada Balsam) are quite expensive. Others can be made in the kitchen from readily available materials (Glycerol jelly).

Ease of use: Here we have to consider two aspects, the preparation of the specimen prior to mounting and the actual mounting process. Some mounting media require the specimens to be dehydrated and fixed before mounting (for resin-based media). This can be a time consuming process. During the mounting process, some media are more prone to form air bubbles (Glycerol jelly).