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Microscopes 
Slides |
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Safety: Microscopy experiments in general rank
on our rating scale; however, certain factors can modify this--
for example, drawing blood, using samples containing pond bacteria, growing
crystals of a toxic reagent, etc. Collecting blood samples presents
no extraordinary hazard as long as sterile instruments are used and care
is taken that students don't expose others to their blood or to contaminated
lancets. Articles, text, and graphics on this website are subject to Copyright.
This page contains three experiments:
Crystal Growth (see below)
Pond Life
Blood Cells
1. Crystal growth under the microscope
This fascinating demonstration touches on the subject of chemical microscopy. It is meant
for biological or light-transmittance microscopes (such as the Observer or the Outfitter).
With some practice at moving the slide steadily, you can actually follow
the edge of the crystal formation as it spreads like feathery tree branches.
Obtain any one of the following chemicals: Magnesium sulfate (epsom
salts), boric acid, magnesium citrate, sodium sulfate, ammonium alum, potassium
alum, chromium potassium sulfate (chrome alum), ferrous ammonium sulfate,
copper sulfate, potassium ferricyanide, ferric chloride, or cupric chloride.
These are of course not the only possibilities- almost anything that crystallizes
can be observed under the microscope. Colorful, transition-metal salts
are of course preferable. Potassium ferricyanide forms beautiful orange
to red crystals. Nickel salts give beautiful, green crystals, but
one needs to respect their toxicity (there is sufficient evidence to class
Ni2+ as a known human carcinogen).
Place a small amount of the chemical of choice (an amount the size of
a match head will suffice) into a spot plate well or a small test tube
and add distilled water to it, one drop at a time, until the chemical dissolves
completely. Add only enough water to dissolve the chemical.
The goal is to produce a saturated or nearly-saturated solution. Safety Reminder: exercise caution appropriate
to the compound used. Magnesium sulfate, magnesium citrate, boric acid, and
alum are relatively harmless, while most of the others present no great danger
if handled with common sense (i.e.,
wash the hands after use; do not ingest or get in the eyes).
Add one or two drops of detergent or liquid soap to the chemical solution
(this is optional). This will greatly lower the surface tension and allow
it to spread thinly across the surface of the slide. It will not prevent
crystallization, however; the evaporation of the water droplet forces crystallization.
Variation: Crush
five or six aspirin tablets and dissolve them in 50 mL of isopropyl alcohol.
Stir and allow the insoluble matter to settle. Place a drop of the
clear solution on a clean microscope slide and observe it under the microsope
as the liquid evaporates.
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| Crystals of acetylsalicylic acid (aspirin)
at 40x, viewed by transmitted light (shined from underneath the stage) |
The same crystals
viewed with reflected light shined from the side. The built-in stage
illuminator is off. |
With a clean dropper or Pasteur pipette, take some of the solution,
being careful not to pick up any undissolved crystals. Place one or two
drops on a clean microscope slide and tilt the slide to make it spread out
thinly. Carefully place the slide on the microscope stage (being careful
not to spill any liquid).
Turn on the microscope's illumination and watch as the water evaporates
(this can take as much as 20 minutes). A thin layer of crystals can be
observed to form at the edge of evaporating liquid. A halogen desk lamp
shining on the slide will cause the evaporation to go much more rapidly
so you can follow the edge of the crystal formation. With colorless compounds
such as magnesium sulfate, altering the angle of incident light can help
make the crystals more visible.
Slower evaporation will produce larger, thicker crystals. A 5 mL micro beaker inverted,
placed over the evaporating drop and left overnight can produce good results.
This experiment is ideally suited to the Mini-VID
eyepiece camera - set one in the eyetube of an Observer III or Observer IV microscope, connect
the camera to a video screen, and a whole classroom can watch.
The shape, color, and optical properties of microscopic crystals can
serve to identify many compounds, both organic and inorganic. This
kind of identification is the primary concern of chemical microscopy.2. Microscopic lifeforms in action
Hay Infusion Microscopy (Microorganisms From Pond Water)
There are two ways one can pursue this. In the first variant,
simply draw some water from the shallow regions of a pond and study it with
a microscope.
The more stagnant the pond, the more microscopic life will be evident.
The best sources will contain hair-like strands of green algae that
are visible to the unaided eye. Try to pipette up some sediment, debris,
and algae strands in your sample jar. Handle pond water carefully,
and do not drink it; some of the species in it may be parasitic and/or
pathogenic.
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Just a few mL of pond water can contain
thousands upon thousands of organisms. The miniature ecosystem in this
micro beaker
was still teeming with live organisms after several days, even without exposure
to sunlight. It is surprising what creatures lurk in the water. Some have peculiar habits. There was a protist we nicknamed "whiplash creature". If you find one, you will know it; unfortunately, they are very difficult to photograph. |
The second variant is a classic experiment from the earliest days of microscopy.
The appearance of pond organisms from seemingly pure water and hay (which
was often found nowhere near a pond) must at first have puzzled early observers
who didn't realize that protozoans could survive pond evaporation to form
cysts that were carried long distances by the wind.
Find some hay, dead grass, or other dry vegetation. Obtain a jar with
an airtight lid or stopper and put the hay into it. Fill the jar about
2/3 full with tap or distilled water, replace the lid, and let it sit at
room temperature for about a week. This "hay infusion" will smell quite
foul and should not be spilled. Wash the hands thoroughly after handling
it. (There are bacteria in this liquid, and though it's unlikely they're
dangerous strains, it is impossible to guarantee this. We cannot control
where you get your water or your hay. Regardless, DO NOT INGEST THE LIQUID and do NOT allow it
to contact eyes, mucous membranes, or cuts on the skin. To accelerate
the process of microbe growth, use pond water instead of tap or distilled
water - although this will spoil things if your intent is to show that the
hay, and not the water, was the source of the microbes. If you're
pursuing the latter course, be sure to sterilize the water and any container(s)
used, leaving the hay as the only possible source of protozoa.
On a clean slide, build a "well" - a square or round, walled-in area
using a thin bead of wax, petrolatum, grease, or quick-drying glue. (You
can also use slides pre-made with a circular
depression). This will hold the drop of water and allow the organisms
to survive for quite some time while viewing under the microscope.
The downside of this is that you will never be able to fixate upon any
particular organism for more than a split second, since they move so rapidly
and will have plenty of space to go. However, if your "hay infusion" has
grown sufficiently busy with protozoans, it will be worth it to prepare
the slide this way.
Place a drop of hay infusion in the "well" that you've created and view
it under fairly low-power magnification (start with 40x, and no more than
perhaps 100x total magnification). View the assortment of protozoans that
dart across the field of view. If your students want a challenge, open
a microbiology textbook with pictures and try having them compare it with
the organisms that dart by. See how many they can identify. There can be
considerable variation in what grows in the infusion, although some common
ones are paramecia, rotifers, stentors, euglenae, and amoebae. Note that
certain protists (such as hydras and certain rotifers) are stationary and
will be found anchored on something (e.g., pieces of pond debris).
If you want to kill everything in the hay infusion (a good idea prior
to disposing of it), don't bother boiling it. This will not kill
all the microbes; cysts in particular may survive boiling.
Strong chlorine bleach, however, should kill nearly everything when left
to stand. 3. Blood cells under the microscope
For years it has been customary to make blood samples by piercing the
fingertip with a sterile needle. Despite fears of blood-borne infection,
a sterilized lancet is certainly safer than the rusty nail on which the
author accidentally cut his hand one day. With this in mind he decided a
tiny pinhole in the fingertip wouldn't be much of an additional threat.
If you do decide to prepare blood samples using the traditional method of
extracting it from your fingertip, make sure you're using a sterile
lancet, and swab the area with alcohol or iodine tincture prior to piercing
the skin. Don't let others come into contact the blood or the used
lancet.
In the present experiment, a blood sample was placed near one end of
a clean slide. The edge of a coverslip was used to smear out the sample
and then the slip was laid down onto the resulting blood smear. No stain
or additive was used for this experiment. Photograph is shown below; it was
taken using an Observer III microscope
fitted with a USB Mini-VID eyepiece camera.
The spiky appearance of some blood cells is common for blood
that's left outside the body. The erythrocytes' normal morphology is destroyed
from drying or aging of the sample, and the cells assume the "echinocyte"
form. The process is known as crenation. There are ways to reduce
the tendency, such as by promptly heat-fixing the smear (110 to 145°
C). 
Photo: © 2003, CR Scientific
Crenation, by the way, can be distressing to the neophyte who first
sees it and has no idea what it's called or what causes it. Beginning
students may look at their crenated erythrocytes and assume they've some
rare, horrible disease with a hyphen in its name. Since psychological
terror is not usually the goal of classroom exercises, it might be wise
to tell students what to expect prior to viewing a blood smear.
If you wish to view leukocytes (white blood cells) and other components
of the blood, it's necessary to use a stain
preparation on your blood sample. There are several different
types of leukocytes, each of which may require a slightly different stain
and staining technique for best results. A commonly-used one is Wright's
stain, which is a combination of eosin Y and aged methylene blue in methanol.
It is good for staining eosinophils. Best results occur if the solution
and sample are buffered at about pH 6.5.
Giemsa stain and Papanicolaou stain are also used for leukocytes.
Nearly any of the methylene blue staining variants (Unna, Nocht, Romanowsky,
Ehrlich) will work for observing white blood cells. In fact, any nuclear
stain should work at least well enough to make the leukocytes visible. When
you do manage to reveal them, you will probably see mostly neutrophils.
Neutrophils are the most abundant white blood cells in human blood; their
nuclei have a distinctive, multi-lobed habit.Back to top
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