Getting Inside an Ant's
Difficulty:
Head
Shawn Carlson, Ph.D.
THE TOY microscope set I got one
childhood Christmas almost put me off
microscopy for good. Squinting through
a cheap plastic eyepiece, manipulating
the sloppy focus control with one hand
and struggling with the other to position
the factory-prepared specimen slides
resulted in more neck aches and
frustration than reward. So years later
when a friend offered me his old
professional-quality microscope for a
mere fraction of its market value, I
hesitated. Besides, I could scarcely see
through the dirt-encrusted optics, and the
gearing for the traveling stage was so
gunked up that it scarcely traveled at all.
But a little patience, along with cleaning
fluid and grease for the gears, quickly
restored this binocular beauty to mint
condition. Today I'm convinced it was
the best $100 I ever spent.
My vintage Spencer has been my
constant companion for more than a
decade. With it I have poked about
inside plant cells, swum abreast of
wriggling sperm and gotten delightfully
lost inside floating forests of
phytoplankton. Unfortunately, no
biological specimen can be enjoyed
under a microscope for long before
natural processes begin to destroy it.
Tissues quickly dry out and shrink, and
bacteria begin breaking down the
delicate structures of life almost
immediately after a specimen dies.
Microscopists therefore routinely
preserve their treasures before
scrutinizing them carefully. The process,
called fixation, replaces the water in the
specimen's tissues with a chemical that
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is incompatible with life. The fluid
pressure keeps the cells plump while the
chemical kills decay-producing bacteria.
Cellular proteins are not soluble in many
fixing agents, however; they precipitate
to form a sort of plaster that sticks to cell
walls. This process stiffens the cell wall
and can actually increase its index of
refraction, making the cell stand out
under the microscope, but it ultimately
destroys potentially useful information.
When citizen scientist
extraordinaire George Schmermund of
Vista, Calif., recently brought together
his interests in insects and microscopy,
he uncovered a wonderful fact. Different
tissues within an insect absorb the fixing
agent he used at different stages in the
fixing process. By observing the
specimen throughout fixation, rather
than waiting until the process is
complete, as microscopists are often
taught to do, he found that separate
organ systems within the animal became
highlighted sequentially. This allows
Schmermund to take the stunning
pictures of insect insides that you see
starting on the next page. His technique
empowers any amateur microscopist to
explore insect interiors in a detail
unmatched by any other method I know.
Begin your own fantastic voyage
by securing some suitable specimens.
Small ants and fleas make ideal subjects.
Schmermund kills them by placing them
in his freezer for a few hours. His fixing
agent is ordinary isopropyl (rubbing)
alcohol. Isopropyl alcohol is
inexpensive, readily available and easy
to handle.
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Figure 1: Getting inside an ant's head is easy. This photo shows that this ant has a brain with a complex double
lobed structure. Note the striking symmetry, and the apparent corpus coliseum (the band of tissue between the two
hemispheres. Photo by George Schmermund.

My local drugstore stocks pure (anhydrous) isopropyl alcohol from

isopropyl alcohol in concentrations of 70
and 91 percent. Buy the highest
concentration you can find. If all you
want to do is take create pictures of inset
insides, this is all you will need.
However, for reasons that will become
clear in a moment, if you intend to create
a permanent library of mounted
specimens you'll also need to purchase
a chemical supply company. (Note that
denatured alcohol is a blend of mostly
ethanol and methanol; it is not isopropyl
alcohol. Both substances are also fixing
agents, but methanol is highly
poisonous. If you choose to experiment
with denatured alcohol, take appropriate
precautions to protect your family and
pets.)

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The box above gives a recipe for
diluting your fixing solution to any
desired concentration. Schmermund took
these photographs using only two
dilutions: 35 and 70 percent. The time
required at each stage depends on the
specimen's size. Soak garden ants and
fleas for at least one hour. Larger insects
may take as long as six hours. The
volume of solution should be at least 20
times that of your specimen's body, but
for ant-size specimens that is still a tiny
amount of alcohol. Schmermund soaks
his specimens in bottle caps and
transfers the chemicals with
eyedroppers.
To get views like those you see
here, you must regularly check the insect
while fixing. Transfer the insect to a well
slide (a slide with a polished depression
to receive the sample) with a drop of
fixing solution, rest a slide cover on top,
then set sail into uncharted waters.
Transfer and position the specimen with
laboratory tweezers (available at almost
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any laboratory supply store and often at
your local swap meet) and an
eyedropper.
Unfortunately, neither water nor
alcohol mixes with the compounds used
to glue your specimen permanently to
the slide. So to share your specimen with
posterity, you must first replace every
trace of both water and alcohol with a
solvent that will mix with the glue. The
process that removes the alcohol is
called clearing. Clearing will make your
specimen largely transparent and will
destroy much of the contrast created
during the fixing stages. You can lock in
some permanent contrast by staining
your specimen with various
commercially available dyes, such as
borax carmine or alum cochineal. But no
method I know of produces the level of
contrast in specific tissues that naturally
develops during the fixing process.
Xylene is the clearing agent of
choice for the amateur scientist. I
recently purchased 32 ounces (a lifetime
supply!) for $5 in the paint section of my
local hardware store. But watch out:
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xylene is poisonous, it dissolves plastic darkroom. Low magnification means
on contact, and its fumes can make you good depth of field. You could do the
quite sick. So be particularly careful to same thing without the darkroom with a
protect yourself and your family and good digital camera.
pets. In addition, although water mixes
with alcohol and alcohol mixes with
xylene, water and xylene do not mix.
The water must be completely removed
with a final fixing step in 100 percent
anhydrous isopropyl alcohol before
clearing.
Once the specimen has been
soaked in pure alcohol for two hours or
more, place it in a solution of equal parts
of xylene and anhydrous isopropyl
alcohol. Cover your container to keep
the xylene from evaporating. Your
specimen will probably float at first. Let
it soak for an hour or two after it sinks to
the bottom and then replace the mixture
with pure xylene. Your specimen should
become fairly translucent at this stage.
Dark patches mark water that was not
removed during fixation. Returning the
specimen to the anhydrous alcohol for
several hours and then re-clearing it can
often clear these up.
Scientific supply companies sell
Canada Balsam and a variety of plastic
resins for the permanent mounting of
specimens. The resin dissolves in
xylene, and so it infuses into the insect's Figure 2: A flea's leg (top) has clearly visible
tissues before hardening. To make your muscles and striations. The mouthparts of a
final slide, place your cleared specimen wood tick are revealed in detail (bottom)
in the depression in the well slide, add a
drop of resin and gently angle the slide Question for Science Fair
cover down on top, being careful not to
trap any air bubbles. Let the resin set Projects
before moving the slide.
One final note. Some If you're doing a science fair project
microscopists have marveled at the you'll need a hypothesis to pose. Here
striking depth of field visible in are a few great ones that can lead you to
Schmermund's handiwork. Here's his championship science fair projects. If
secret. These photographs were taken none of these appeal to you, that's fine.
with 35-millimeter film at low Ask your own questions. Doing what
magnification and then enlarged in a interests you is the always the best way
to approach any science project.

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 Question One: How does an
ant's brain size depend on its body size?
Secure about ten specimens of several
ant species, and use your microscope to
measure the size of the ant as well as the
size of its brain. Take an average for
members of the same species and plot
the average of different species as brain
size (Y axis) vs. body size (X axis). (The
best way to measure size is with an
eyepiece that has a ruled pattern [called
a reticule] printed on it. But you can also
do it by putting a standard object, like a
BB next to the ant and estimating the
size of the insect in terms of how many
BBs long it is.)
Can you come up with a simple
measure (perhaps the width of the
thorax) that correlates to body size? Can
you prove that it is a good measure? If
so, then you only have to measure this
one simple parameter to estimate the
ants body size. What other questions
does all this suggest to you?
Question Two: Does the queen
ant have a brain structure that is in any
way different from the workers? What
about the male drowns and other ant
specialists? Could different behavior in
different specialists within of an ant
colony show up different degrees of
development of the separate brain
structures? Does the brain of an old
queen look any different from the brain
of a new queen?
Resources
You don't have to search the Web for the
materials you need to do this project. I
have has already done all the hard work
for you! Just go to www.scifair.org and
follow the product links "Marvelous
Microscopy." There you will find book
and product reviews together with links
to companies that offer the best deals on
everything you need. This is a FREE
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service and part of any purchase you
make will support citizen science and
science literacy.
Organizations
The Society for Amateur Scientists
(SAS) is a 501 (c)(3) nonprofit
organization dedicated to helping people
enrich their lives by following their
passion to take part in scientific
adventures of all kinds. All amateur
scientists should be members of this
unique and wonderful organization.
Check them out at w w w . s a s . o r g ,
especially their E-sine, The Citizen
Scientist and LABRats.
Scifair.org is a free Web site
maintained by the author and SAS to
help you with your science fair projects.
Surf over to www.scifair.org to take
advantage of the services it offers.
About the Author
Shawn Carlson, Ph.D. (Dr. Shawn) is a
physicist and MacArthur Fellow, and is
the Founder and Executive Director of
the Society for Amateur Scientists and
the inventor of LABRats. He wrote "The
Amateur Scientist" column for Scientific
American magazine for the last six of the
feature's 73-year run. If you like this
project, check out Dr. Shawn's monster
CD-ROM collection with over one
thousand extraordinary science projects
from all fields of science. It is available
at www.brightscience.com
NOTICE
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