There is a long tradition in microscopy of a blending of scientific and aesthetic perspectives. Almost every microscopist has had a particular group or type of specimen to which he or she has a special partiality. For some, it is the marvel of micro-crystals under polarization; for others, it is diatoms or desmids, or foraminifera or radiolaria. It's not really necessary to create a hierarchical ranking; each of these types of specimens has its own unique charm and interest. Certainly, no one can deny that radiolaria are among the most beautiful and intricate denizens of the microworld.

Radiolaria shells. Darkfield illumination.
Image by Wim van Egmond.

From a cursory examination of bibliographies, I am under the impression that the literature on radiolaria is rather limited compared to the literature on forams and diatoms. There certainly doesn't seem to be any convenient reference that provides a good survey for the amateur, which means that getting a sense of the taxonomy of this group is very difficult. There is an excellent technical book simply called Radiolaria by O. Roger Anderson published by Springer Verlag, but its current price is $205.00!! If they sold it for a quarter of that price, they probably would sell ten times as many. Springer is notorious for charging outrageous prices for technical books and having them show up a few years later remaindered for a tenth or even a twentieth of their original prices. Seems to me a funny way to run a business. Hey, is anybody from Springer out there listening?

All of this is further complicated by the fact that many of the forms are known only by their shells and this is true of both fossil and extant species. To make matters even more interesting, the shells of some species show some morphological variation and furthermore most samples are processed in such a manner that a significant proportion of the specimens are chipped or broken and very often this involves the loss of spines and other structures which may be crucial in identification.

In this discussion, I will limit myself to dealing with shells from "radiolarian ooze." Dealing with living, planktonic forms is entirely another matter and one which, unfortunately, I know nothing about.

So, where do we start? The first thing is to purchase a small vial of cleaned shells from "radiolarian ooze." This ooze can be several hundred feet thick at certain spots on the ocean floor. It is not so easy to dredge up and not easy to clean, so the samples are moderately expensive. When you get your vial from a biological supply house, your initial reaction on examining it may be shock—What? I just paid $20 for a half inch of sand in the bottom of this little glass tube? But rest assured, you have made a wonderful investment; that tiny sample will contain thousands and thousands of elegant glass (silica) shells.

The sample may be stored in distilled water, water and glycerin, or alcohol. If you wish to examine the shells in a liquid medium, the distilled water is preferable. Don't use tap water or artesian water as these contain salts which can form deposits on the shells if you decide to let them dry for dry mounting or they can cause annoying precipitates in mounting media.

A classical problem in mounting radiolaria (and diatoms) is their transparency as a consequence of their siliceous composition. If you use a standard mounting medium, then you are looking through glass lenses, glass prisms, a glass coverslip, a glass slide and more glass in the condenser which is providing the illumination for these tiny glass shells mounted in a substance that has a refractive index very close to that of glass—and so, the radiolaria become virtually invisible. The traditional solution has been to mount such specimens in a medium with a considerably higher refractive index than glass. This approach has several disadvantages. Some of the best media are now virtually unobtainable. Most available media are very expensive and frequently present a greater problem with air bubbles forming around the specimen than do standard media. Worst of all, some media that produce the highest refractive indices, such as realgar, involve extremely dangerous techniques that should never be attempted by the amateur.

Almost every type of specimen presents special challenges in order to view it under optimal conditions, but radiolaria are among the most difficult. As we have already noted, the fact that they are glass presents special obstacles and even though diatoms are similarly constituted, they are generally thinner and, as a consequence, do not present the same degree of difficulty in terms of depth of field. My recommendation is that if you want mounts of radiolaria in a medium with a high refractive index, purchase them from a biological supply house which produces high quality preparations. These are relatively inexpensive and a good addition to one's collection of permanent slides.

For making my own preparations of radiolaria, I decided on a quite different approach. It occurred to me that if I could find a means of coating them, then I could use oblique illumination and also not have to worry about having a mounting medium with a high refractive index So, the first task was to find some kind of stain or coating which would provide the right sort of contrast. Attempting to stain silica using biological stains would, I knew, be hopeless; however, I thought that perhaps a thick, but not too thick solution of stain, might deposit a thin film and provide the right sort of contrast. I tried Malachite Green, Alizarin Red S, Orange G, Nigrosin, and Neutral Red—and the results were either no staining or a sort of sticky mess that obscured the structural details of the shells. Finally I decided to try out the silver staining method that I had developed for forams. I have already described this technique in another article titled: Silverizing Forams; however, I will give a brief summary here as it applies to radiolaria.

It is imperative that the specimens be clean and free from salt deposits. Put a sample in the bottom of a small vial and vigorously add distilled water using a pipet or wash bottle. This will agitate the tiny shells in the water helping to remove any deposits. Allow the sample to settle for about an hour and then using a micro-pipet, carefully remove most of the water. Repeat this process several times always being sure to allow sufficient time for the shells to settle back to the bottom. Transfer a minute sample of the shells to a clean slide and, when dry, examine them under the microscope. If the shells are till not clean, it may be that there is an alkaline deposit. This can generally be removed by treating the sample with dilute acetic acid in the manner in which the washing with distilled water was done. After the treatment with acid, it is then necessary to rinse the sample several times with distilled water again.

Once the shells are clean, we can begin the staining procedure. Use a small, shallow glass or plastic dish and transfer a small sample of the shells to one edge of the dish in as little water as possible. Add 2 or 3 drops of 1% silver nitrate solution (This can usually be made up by a local pharmacy) and allow the specimens to sit for 5 to 10 minutes or even longer. Time is not crucial to this part of the procedure.

Prepare a Vitamin C solution by dissolving a 1000 mg. tablet in 60 cc. of distilled water. Add 2 or 3 drops of this to the shells in the dish. At this point, timing becomes important. The specimens will begin to turn brown as the silver begins to precipitate and then a gray film will start to form on the surface of the fluid. About 2 minutes after the gray film forms, remove as much liquid as possible using a micro-pipet. Next squirt 90% alcohol over the radiolaria. This helps remove undesirable clumps of silver particles from the specimens, but without removing the tiny layer which we want, since silver nitrate while readily soluble in water, is only very slightly soluble in alcohol.

Transfer a small drop of radiolaria to a clean slide and allow the alcohol to evaporate. Add a drop of xylene, position the radiolaria and then add the mounting medium. (Caution: Xylene is poisonous and highly flammable.) The silver stain produces very good contrast and specimens stained in this manner are best observed with oblique top illumination.

A Note Regarding Silver Nitrate

All metal salts should be handled with caution and silver nitrate is no exception. Both the crystals and the solutions are poisonous and caustic. Silver nitrate is also highly reactive and should not be mixed with chemicals other than those specified.

Comments to the author Richard Howey welcomed.

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

Related articles. Also see Brian Darnton's and Mike Samworth's articles on aspects of the radiolaria.

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Published in the August 1999 edition of Micscape Magazine.

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