Permanent Slides: Pros and Cons |
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 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'.
Published in March 1999 Micscape Magazine.
Please report any Web problems
or offer general comments to the Micscape Editor,
via the contact on current Micscape Index.
Micscape is the on-line monthly
magazine of the Microscopy UK web
site at Microscopy-UK