An overview of the specimen preparation steps and choice of mounting medium for making permanent slides for microscopy.
What are permanent slides: Permanent slides carry specimens that are preserved and mounted in mounting medium. They can be kept for a long time. Permanent slides can be bought or they can be made at home. Some people are perfectly satisfied with observing commercially prepared slides. Others find much enjoyment in observing specimens of their immediate environment and in preparing their own permanent slides. Permanent slides use a mounting medium that becomes solid (other possibilities exist as well, such as dry-mounted slides). The specimen is preserved and if properly made, the slide can withstand a century and still be usable.
Commercial permanent slides
Rare specimens: Commercially prepared permanent slides can be bought online and/or from school supplies companies. Sometimes microscopes already come with a set of permanent slides. They allow you to observe specimens which are otherwise difficult if not impossible to obtain. External parasites of animals (fleas), animal tissue sections, cell division stages, etc. are all interesting to look at. These are, however, difficult to make for inexperienced beginners, especially if the appropriate laboratory practice and materials are missing. Larger tissue parts have to be fixed (for preservation), embedded and then stained and microtomed (cut into thin sections). I therefore recommend that you buy yourself a box of 25 or 50 prepared slides in any case.
Sometimes elaborate preparation: The preparation of a permanent slide can be (dependent on the specimen) an elaborate and time-consuming process. Proper specimen preparation is really important here. The specimen must first be fixed before it can be embedded into mounting medium. Otherwise it will not be stable and might decompose over time. It might also be necessary to cut the specimen into an appropriate size (microtoming). This can be quite specimen specific and may require you to read some literature. In many cases amateur microscopists do not have the equipment, chemicals, lab (or interest!) to prepare a specimen for only a handful of permanent slides. If this is the case, then I recommend that you buy a box of permanent slides with interesting specimens for direct observation.
Because it is possible these days make photographs of the specimens under the microscope, the “need” to make permanent slides is probably lower than it was before. But I think that this might be the wrong question anyway. If it interests you and if you enjoy a little bit of lab work, then why not give it a try.
Preparing the specimen
Before the specimen can be permanently mounted, it must be preserved and also made small enough to fit on a microscope slide. Specimen preparation might include all (or only some) of the step below. It all depends on the individual specimen.
- Fixing: This kills the cells and preserves the specimen. The fixing solution is water-based and a wide range of different solutions exist. Sometimes this step can be omitted, if the specimen is not alive, or if there are no cells present. This includes animal fur, for example.
- Staining: The specimen is stained so make certain structure better visible. This can be done by placing it into the stain for several days.
- Dehydration: The water in the specimen is slowly removed by placing it into subsequently higher concentrations of alcohol. The slow increase in alcohol concentration prevents that the specimen shrinks. Again, not all specimens require this step, because they are already dry. Other specimens (such as protozoa and algae) are very sensitive to dehydration and therefore they are not completely dehydrated (you use glycerine jelly, which contains water).
- Infiltration: The alcohol is removed and replaced with a solvent which is compatible to the embedding medium (usually paraffin). Often xylene is used. This step also depends much on the mounting medium to be used, because the solvent has to be compatible. Many amateurs try to completely avoid these solvents due to safety concerns.
- Embedding: The specimen is now embedded into paraffin. The paraffin block supports the specimen during microtoming. This is the case for soft specimens, such as animal tissue. The paraffin provides appropriate support.
- Mirotoming: The specimen (in paraffin) is now cut into thin sections with the help of a microtome. Some plant tissues can be directly microtomed without embedding.
- Mounting: The paraffin has to be removed and the specimen is then mounted in appropriate mounting medium.
Large samples of tissue, for example, have to be first fixed for preservation. The tissue is first placed for several days into an appropriate fixing solution. This will kill the cells and “freeze” them in time. This solution is often water-based. This water has to be removed before further preparation. The tissue sample is dehydrated by placing it into subsequently higher concentrations of alcohol. Dropping the tissue into concentrated alcohol will cause rapid shrinking, and bad results. Finally, the alcohol has to be removed and the specimen is placed into xylene, which is not healthy when inhaled. Now the tissue is embedded in paraffin wax so that it can be sliced into thin sections with the help of a microtome. The wax has to be removed and the thin sections can now be mounted in appropriate mounting medium. You then have to wait for a few weeks for the medium to dry. Oh, I forgot: You also might want to stain the sample….
Making easy permanent mounts
Choose dry specimens: You want to make your own collection of permanent slides, but do not want to get too involved in preparing the specimen? There is good news. Making a permanent slide can be as easy as making a wet-mount, provided that you choose the correct specimen. Specimens which are already sufficiently small and dry can be easily processed into permanent slides. Dry insect parts, such as wings or legs, animal fur, wood scrapings, etc can be directly mounted. These specimens do not shrink and deform too much when drying. It is also not necessary to cut them into sections with a microtome. Place a drop of mounting medium on the slide, then place the dry specimen into the medium. Add another small drop on top, then add the cover glass. You still have to wait until the mounting medium has dried. Store the slide horizontally in a well-ventilated area. To give you an example: Over the last few years I have been bitten by a tick several times. Every tick that I pulled out of my skin was briefly placed into alcohol (for water removal) and then mounted in Euparal.
Try nail polish: One of my favorite mounting media is Euparal. This is also a preferred mounting medium for preparing insects. Entomologists (insect scientists) like to use it a lot. Unfortunately it is not always easy to obtain. If you just want to experiment making slides, then I recommend that you first try clear nail polish as a mounting medium. Nail polish has the disadvantage that it shrinks quite a bit when drying, and bubbles can form beneath the cover glass. I therefore recommend that you allow the solvent of the nail polish to evaporate a little before placing the cover glass on top.
Choice of the mounting medium
Types of mounting media: Mounting media can be classified into aqueous (water based) mounting media and solvent based media. Specimens that are to be mounted in solvent based mounting media must be completely free of water. A common water-based mounting medium is Glycerine gelatin. This medium can easily be made at home, but is difficult to use. Bubbles are common and the slide has to be warmed during the mounting process to keep the medium fluid. Solvent based mounting media sometimes contain harmful organic solvents, which might make these media less suitable for amateur use.
Choice of mounting medium: There are numerous different mounting media available for making permanent slides. What factors determine the choice of the mounting medium? Here are some possible points to consider.
- 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 antiseptics. This aspect of toxicity is something to consider when making permanent mounts either as a hobby or for educational purposes in schools.
- 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.
- Compatibility with specimen: Specimes which are kept in water should be transferred into a water-based mounting medium (eg. algae into gycerine jelly). 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. Some modern mounting media are fast drying (used for quick tissue preparation in medicine) but the durability is lower. If you want you slides to be usable after a century, use slow-drying mounting media that will soak into all parts of the specimen.
- Cost and availability: Some mounting media (such as Canada Balsam) are quite expensive. Others can be made in the kitchen from readily available materials (Glycerol jelly), others are readily available in the local supermarket (clear nail polish). Yet others are available only over laboratory supplies companies that do not ship to private people.
- 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).
What if the specimen and mounting medium don’t match?
Compatibility issue: This clouding is due to an incompatibility between the specimen and the mounting medium. This happens when a non-water based mounting medium (such as Canada Balsam, Euparal or other resin based media) is used with an improperly dehydrated specimen. The water from the specimen is driven out by the solvents of the mounting medium but are not able to mix with the mounting medium, forming bubbles.
If Euparal is used as a mounting medium, then it is possible to transfer the specimen directly from alcohol into the mounting medium. If other mounting media are used (Canada Balsam, etc), then it is necessary to infiltrate the specimen first in xylene before mounting. Be aware of health risks when inhaling these solvents!
Specimen stability: Mounting a moist specimen with a non-water based mounting medium is also problematic because the specimen is not properly preserved. The mounting medium prevents a drying of the specimen. Bacterial decomposition can then start to take place.
Prevention: A clouding can be prevented by first dehydrating the specimen. This can either be done in air (for those that do not shrink) or by placing it into subsequently higher concentrations of alcohol. The step-wise increase in alcohol concentration slowly withdraws water, reducing the possibility of specimen shrinkage. various concentrations of ethyl alcohol (ethanol) can be used for this. As ethanol itself also contains small amounts of water (from air moisture), it is sometimes recommended to use isopropyl alcohol (isopropanol) for the last dehydration step. This alcohol is “dryer” than ethanol as it has a lower tendency to attract air moisture.