Permanent Slides: Pros and Cons

by Richard L. Howey, Wyoming, US

 

Werner Nachtigall in his book Exploring With The Microscope (an excellent book for the beginner and amateur and it contains many splendid color photomicrographs) makes a rather radical assertion: "The only permanent slides that I can recommend with good conscience that students make themselves are diatoms." (p. 80) While I have considerable sympathy for Dr. Nachtigall's sentiments, I also have a number of reservations.

As a professional biologist, Dr. Nachtigall has access not only to equipment, but to many supplies and kinds of apparatus not readily available to the amateur or hobbyist microscopist. As a consequence, being a professional, he may be setting standards which are not always appropriate to the amateur who can learn a great deal by experimenting with making certain types of permanent slides. On the previous page, he makes a persuasive argument for his position when he says: "Some companies make permanent slides that are expertly prepared, perfectly stained, extremely thin, safely embedded, and they cost very little. What more can anyone possibly want?" To my mind, this certainly includes diatom slides. Occasionally one can collect rich concentrated samples of diatoms, but in my experience, this is the exception rather than the rule. The classical procedures for separating diatoms from surrounding detritus are tedious, and the techniques for cleaning them are not only time-consuming, but involve the use of powerful and dangerous chemicals which must be handled with extreme caution and are certainly not appropriate for the average amateur. Furthermore, to get "expertly prepared" mounts of diatoms, i.e., ones in which one can see the fine detail of the frustules, it is necessary to use mountants with a high refractive index and these mountants are often difficult to obtain and/or are very expensive. However, especially for the larger diatoms, if one is not needful of fine detail for taxonomic purposes, then Dr. Nachtigall's suggestions for cleaning and mounting diatoms are quite appropriate.

The reason that refractive index is such a problem with diatom frustules is that they are composed of silica (that is, they are essentially glass). So, you are using glass lenses to look at tiny glass shells sandwiched between a glass coverslip and a glass slide. As a consequence, contrast and resolution become crucial difficulties. There are other less popular, but no less intriguing, objects which have the advantage of being calcareous and thus of a lower refractive index. So, I want to use this essay to encourage amateurs to make permanent slides of these kinds of objects and a variety of other objects as well. However, let me state clearly that I strongly agree with Dr. Nachtigall's assertion that nearly all amateurs should not expend a lot of effort trying to make permanent mounts which involve sections of plant or animal tissue. Protozoa, with the exception of shells, rarely mount well. Small worms and other highly contractile organisms are also not good candidates, although there are some interesting exceptions.

One of my favorite types of calcareous objects to mount is sea cucumber spicules. Not all sea cucumbers have spicules, but most do and frequently they occur in great numbers just below the surface of the skin. I take a sharp scalpel and cut a few small, thin sections of the outer layer and place them in a test tube containing a 10% solution of sodium hypochlorite (household bleach—I hate the smell of this stuff, so I buy the lemon-scented variety which is somewhat less noxious). Plug the tube with cotton as this will allow the accumulation of oxygen to escape as the chemical acts on the tissue. Let the sections soak for 24 hours and then examine to see if all of the tissue has dissolved. If not, carefully pipet off most of the solution and discard it being careful not to disturb the deposit on the bottom of the tube. Add fresh sodium hypochlorite and examine after another 24 hours. Continue this process until only spicules remain in the tube. Pipet off the solution and add distilled water which you can purchase inexpensively by the gallon at your local supermarket. Let the spicules settle to the bottom and then repeat this process a number of times to be sure that all of the sodium hypochlorite has been removed. To check this, take a drop of the solution containing a few spicules, put it on a slide and let the drop evaporate. If, under the microscope, you find that crystals have formed on the slide, then you need to continue the washing process. Once they are clean, they can be stored indefinitely in a tightly sealed vial of distilled water.

Now, after all that work, the question is: What have you got? Well, it depends on the sea cucumber. Some spicules are flat irregularly-shaped plates with circular holes in them, others are very regular and look rather like very simple stick and ball models used in chemistry classes, still others are shaped like miniature anchors, and then there are the elegant spoked wheels of Chirodota which occur in small clumps just below the skin's surface and, to the naked eye, look like accretions of sand grains. And, as if this weren't enough, almost all of these structures are birefringent, that is, under polarized light they show up in a variety of pastel colors. Certain species can be readily identified from their spicules.

When you are ready to make a slide, take a drop of the spicule sediment and place it on a clean slide. You don't want the spicules in clumps, so take a fine needle or bristle from a brush and use it to spread the spicules out over the area for your cover glass. Such a bit of equipment is readily made by trimming a small insect mounting pin, a brush bristle, or even one of your own eyelashes and then mounting it in a wooden matchstick or small dowel. These implements are handy for a variety of microtechniques.

With these spicules, you rarely run into the problem of air bubbles, but it is often best to place a small drop of xylol (or whatever the solvent for the mounting medium is) on the spicules, check their position and then add the mountant and a cover glass. Whenever you are using one of these solvents, exercise great care—they are highly flammable and the vapors can be toxic, so always work in an area that is well-ventilated. It is best to use a synthetic mounting medium and you must make certain that it is of neutral pH. If it is acidic, as balsam is, then sooner or later the mountant will dissolve the spicules and you will have little left to show for all of your work.

On a moment's reflection, you will realize that there is a wide range of objects that dry quite well and lend themselves to mounting with the technique described above. The spines of sand dollars and of small brittle stars can be of considerable interest.

If you are a gastronome with a taste for gastropods and have some extra escargots and aren't squeamish, you can give them the Mary Queen of Scots treatment and then pop their heads into some sodium hypochlorite solution. After the proper treatment, you will discover a long thin band with rows and rows of minute teeth. This is the famous snail's "tongue" or radula. These remarkable structures are sometimes more than half as long as the snail's entire shell. This extraordinary device is what the snail uses to collect its food and the results of its activity can be seen on the sides of aquaria. The teeth of the radula, like those on a woodworking file, scrape the algae off the glass sides leaving a clear, thin trail. Sometimes the radulae are of such size that it is preferable to cut them into several pieces for mounting. There is considerable variety in these structures just as in the different species of aquatic and terrestrial snails, but one should also look at the radulae of garden slugs, limpets, chitons, and even sea hares. With a bit of practice, you can produce very nice mounts and gradually build an impressive collection.

As a microscopist, you, by definition, have a taste for the Lilliputian and the Lepidoptera provide us with dazzling specimens. Don't assume that because you've seen one moth or butterfly scale, you've seen them all—there is a rich variety of shape and color. Scrape some scales onto a slide, distribute them evenly, add a drop of solvent, let it evaporate, then add the mountant and a cover glass. Antennae can be mounted in a similar fashion.

Hairs of various animals also mount easily. The solvent is important in order to dissolve out oils that may be in the hairs. In fact, it is probably best to rinse the hairs in a drop or two of solvent, transfer them to a fresh slide, arrange them, and add mountant. Again, if you examine them closely, you will find a surprising variety. You can also mount various types of synthetic fibers and compare and contrast them both with each other and with various types of hairs.

Feathers are a minor miracle and should first be examined carefully with a stereo dissecting microscope. Even feathers which appear drab to the naked eye can be full of surprises and duck, pheasant, and peacock feathers are glorious. Do me a favor and save the peacock feather for last. I know, I know, it's like opening a fresh box of candy and telling you to save the pecan coconut truffle in white chocolate until the last, but do try.

When observing feathers with the stereo dissecting microscope, play with the illumination. Note very carefully how changes in the angle of light can affect some colors. Here is an opportunity to observe a remarkable phenomenon. Some colors are true pigments, whereas others are "structural" colors, that is, parts of the feather function as optical elements and refract different wavelengths depending upon the angle of illumination, thus producing different colors as the light changes. It is generally best to soak small bits of feather in solvent before mounting to dissolve out any residual oils.

But let's go back to spicules again. I wasn't quite through talking about them. Sponges provide an impressive variety of types from long slender needles to tiny spiky spheres that look like the ball at the end of the chain of a medieval mace. Some sponges, such as the common bath sponge, have no spicules at all and some have calcareous spicules while others have siliceous ones. Collect bits of soft corals or buy some small pieces in a pet store or specialty shop. When you dissolve away the tissue, and by the way pieces that are dry, work perfectly fine) you will be left with a small mound consisting of countless spicules. I have taken small sections of Renilla (the Sea Pansy) and treated them and the pigment in the spicules is so well fixed that even the sodium hypochlorite doesn't bleach it all out and so they remain a beautiful, rich claret color.

The wings and some appendages of small insects can be dried, treated with solvent and mounted. Fish scales make excellent specimens as well, Almost any material that dries well can be mounted. The really difficult materials to mount properly (if it can be done at all) are sections of plant and animal tissue and small delicate organisms, such as protozoa, rotifers, flatworms, etc. It is with these types of specimens that it is generally best to buy professionally prepared slides. However, some of these can be disappointing, more as a consequence of the nature of the specimen itself rather than a lack of skill on the part of the preparator.

Material that has to be dehydrated through a series of alcohols and then cleared, and in many instances sectioned by microtome, usually involves resources beyond the reach of the amateur microscopist.

But, as always, there are exceptions and here we need to shift gears in terms of microtechnique. Ordinarily when one thinks of permanent mounts, one thinks of Canada Balsam or a synthetic resin. However, for nearly a century and a half, microscopists have experimented with alternative mountants. Glycerine, glycerine jelly, and mountants utilizing water-soluble gums, such as gum arabic (acacia), have been fairly widely employed to mount delicate specimens which would be damaged by attempting to dehydrate them by running them through the alcohol series. These types of mountants can give very good results, but are rather messy to work with and demand considerable practice to get satisfactory results.

Fortunately, there is yet another option, namely, a group of media consisting of partial combinations of lactic acid, phenol (carbolic acid), polyvinyl alcohol, glycerine, and water (sometimes with small amounts of other chemicals as well). It is best by far to purchase one of these types of media from a biological supply house if possible and preferably a medium not containing phenol which is very toxic and caustic.

Years ago I bought such a medium from a biological supply company in the U.S. called Turtox which is now defunct. The medium came in two forms: CMC-10 which was clear and CMC-S which was red, due to the addition of Acid Fuchsin for staining the specimens. I bought a pint of each and for a long time, it went unused because I was working almost exclusively on protozoa and did not attempt making permanent mounts. However, in the last two years or so, I have been using it for a variety of specimens and have nearly exhausted my supply.

This medium does not contain phenol (the odor of which is distinctive), but it does contain an acid (probably lactic acid) and is thus not suitable for mounting any type of calcareous materials. This medium also acts as a clearing agent, and in some specimens, increases the visibility of internal structure.

Specimens can be mounted in it directly from water or weak alcohols. I generally rinse specimens in distilled water before mounting to minimize the possibility of crystals forming in the mount as a consequence of residues from the fixative. The mountant acts as a killing agent and so some types of specimens can be placed live on a slide and fixed by means of the mountant. Here again, it is best to carefully rinse the specimen in distilled water, since pond water contains inorganic salts that can crystallize in the medium thus spoiling the preparation.

Small aquatic annelids, loricate rotifers, tardigrades, water mites, siliceous spicules, diatoms, desmids, gastrotrichs, fish scales, bits of feather, butterfly scales, wings of small insects, and many other types of specimens can be mounted quickly and relatively simply in this type of mountant. As with virtually all mounting media, one has to work carefully to minimize the inclusion of air bubbles. Although these procedures are straightforward and can be carried out quickly, this is not an excuse to make sloppy preparations. With a little practice and a bit of care, you can produce quite respectable slides.

Will they be permanent? Well, that's a relative matter. Balsam and synthetic resin mounts could last a very long time. There are, after all, insects that are millions of years old, preserved in amber which is a natural resin. Frankly, I'm not very concerned about whether or not my slides last ten million years. If they last another 50 years, they will surely outlast me and by that time Emperor Bill Gates will probably have outlawed microscopes.

We still haven't yet said much about why one might want to have a collection of permanent slides which one has made oneself. Actually, there are a number of reasons. Sometimes, I come across an unusual specimen that I've not seen before and would like to have a slide of it as a reference to examine in greater detail at a later time. Such slides are valuable in at least two other respects as well: 1) to show to others to illustrate particular types of specimens that might otherwise not be readily available at the moment and 2) to show others to get help in identifying unknown specimens.

If one develops a special interest; for example, types of spicules, then over a period of years, one can gradually build an interesting and important reference collection.

We haven't discussed making liquid or dry or glycerine jelly mounts here, but that will have to wait for another time, as this essay is already too long.

If anyone knows the formula for the Turtox medium CMC-10 and/or CMC-S and if they are in the public domain, I would very much appreciate learning their composition. Also, if anyone knows of a medium of this type which is commercially available, I would be very grateful for the name of the distributors and their addresses.

Comments to the author Comments to the author sent via our contacts page quoting page url plus : ('rhowey','')">Richard Howey welcomed.

Editor's note: The author's other essays on-line can be found by typing in 'Howey' in the search engine of the Article Library, link below.

Click here to read a Micscape review of Nachtigall's book 'Exploring with the Microscope'.

 

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