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Microscope slides
Slides
CR Scientific

Safety:  Handling glass slides and coverslips does present some hazard, but microscopy in general is not an especially dangerous activity.  However, certain factors can modify the hazard level upward-- 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.

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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 Student Advanced ). 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.



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 a biological 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.


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.  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). 
We had advised to boil the water and then add bleach, or just add bleach;  the thinking was that some spores and cysts survive boiling.  Upon further consideration, it seems probable that those few organisms able to survive extended boiling are also going to be the ones immune to ordinary chemicals.  Since the goal here is taking away the contact infection hazard, complete sterilization is not necessary.  Boiling, perhaps with a couple mL of chlorine bleach-- use ventilation!-- will render the hay infusion suitable for disposal.  Since, of course, we know some readers are just waiting for the excuse, there's always the possibility of evaporating the residue and charring it to ash in a mini kiln.
 





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).  


Blood sample viewed through microscope
Photo: 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.


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