Examining pond water samples under low magnification on the microscope is a great way to discover the diversity and amazing adaptations exhibited by micro-organisms.

Here's an example: The tiny (60µ) ciliated protist Halteria is common in stagnant pond water, yet little has been reported about its remarkable behaviors. This pot-shaped cell has a number of long flexible cirri that allow it to spring through the water. I have studied and photographed Halteria for many years and have often wondered what survival advantages are associated with their bouncing behavior. We have the large blue Stentor (400µ) living in our fish pond, so during one filming session, I decided to see if these huge cells would feed on Halteria. A Stentor will quickly settle down on a microscope slide and begin feeding on just about anything its powerful, cilia-driven feeding currents bring near the oral groove. Using a micro-pipette, I introduced about a dozen Halteria, and began recording the scene on video. As the little swarm of Halteria was drawn toward the Stentor's gaping maw, the cells went into a “bouncing frenzy” continuing this extreme outpouring of energy for about ten seconds as, one by one, they shot off to the side away from Stentor's powerful intake flow. The video observation is shown in our video and DVD on protists.

Halteria's bouncing getaway

Wild microorganisms, do we really know them?

Micro-organisms represent one of the most overlooked areas of biology. For example, biologists have identified less than 10% of the bacteria that occur naturally in soils, ponds and oceans. Protists are also relatively unstudied. New protists are discovered and named on a regular basis, and their evolutionary relationships are just beginning to be unraveled.

Bacteria and small amoeba from a sample of pond scum.(Based on shape alone, how many kinds can be found in this video frame?)

Techniques for sampling micro-life communities

Collecting living things from nature has become taboo in many places. We no longer invade tide pools collecting everything in sight, often to have the organisms die in our buckets before we get them into the lab. However, microorganisms are something else. A jar of water removed from a pond does no harm, and can provide a spell-binding safari into unknown territory, where all kinds of personal discoveries are possible. (such as Halteria's bouncing escape).

On a microlife hunt, it's important to recognize that a pond, lake or wetland contains a variety of habitats, each home to a different community of living things. Here is an example of the various collections you might take at one pond:

Clumps of green Stentor on decomposing leaf

Green Stentor. The color is due to symbiotic algae.

You can also set traps for microorganisms. One technique that works quite well is the sponge trap. Cut a cube of open cell dish washing sponge (not used), and spear it on a long sharp stick (barbecue skewers are perfect). Tie a marker flag to the other end (you will need it in a week when you pick up the trap). Set up the sponge traps anywhere you think there might be protists, and make a map of your trap line. We usually leave traps out for a week, but you might try setting up an experiment using different time intervals and possibly discover how microlife communities develop when colonizing a new substrate.

After you collect the sponge traps, squeeze a few drops onto a microscope slide or culture dish and take a peek.

You can also bait for protists. A plastic cup containing a few pieces of dry pet food covered with a nylon stocking held on with a rubber band will often attract large ciliates such as Spirostomum, Paramecium and Vorticella. Just remember where you submerged the cup and be sure to retrieve it.

Collection Jars

Care of your collected samples

Keep your collections cool. If you are on an overnight microlife expedition, a drink cooler with some ice will let you “bring um back alive.” (cooling reduces activity and lowers oxygen demand)

Keep your collections near a window, but away from direct sunlight and sample them regularly. Start with a stereo dissecting scope, set up for transmitted light. Look for interesting protists, rotifers, worms, and other discoveries. Work with the plankton jar right away, as planktonic organisms are often the most likely to die when taken from their open water environment.

More Collection Techniques

Afraid of the water? You don't have to go near it. Find a dried up road side ditch, pond or puddle. Collect a dry mud chip. Add water. Protists do exceedingly well in temporary environments due to their ability to form cysts. Amoebas have crawled out of cysts trapped in Egytian mummy wrappings, attesting to their longevity. Collect a hand full of dried algae or mud chips from the shore of a lowering pond. Within hours after wetting, I find that all sorts of protists have excysted and are cruising around looking for something to eat.

Lake Mud

Dried moss clumps are another source of protists adapted for a quick session of feeding and reproduction, and then back into dormancy awaiting the next rain. Sponge up water in your moss and squeeze it out in a petri dish. In a few hours you may find protists, rotifers, nematodes, and waterbears.

Protists and pollution

You can make a crude judgment about how much organic pollution is present in a body of water by judging the degree of biodiversity. More organisms but fewer species suggests an environment overloaded with nutrients. This is the typical situation where sewage gets into a stream or lake. A similar organic input occurs in runoff from heavily fertilized farm fields. Probably the most extreme cases are found in rain pools near cattle feed lots where cow manure covers the ground. In a rain pool near a particularly smelly feed lot we found an almost pure culture of Euglena. Without competition, and with an unlimited supply of nutrients, the Euglena population simply went wild turning the water pea soup green. Unpolluted natural communities tend to have many species but not necessarily any one species in great number.

Culturing protists

Now that you have several jar collections on a shaded window ledge in your classroom lab you may want to amplify the populations of protists. If bacteria feeders are your choice, add something that will speed up bacteria growth. Try dried lawn clippings, shake in some tropical fish food, or add a chunk of dried pet food. Following this handout, the water turns cloudy with bacteria and withing a few days strings of suspended protists show up, harvesting the bacteria. Termite symbionts

Very few people object to the killing of termites, the destructive wood devouring insects that destroy structures. These very same insects offer some terrific observational adventures. Turn over boards or logs that have been partially buried in damp ground. You may find the wood's surface to be swarming with subterranean termites. If not, split the board with an ax, opening up termite galleries and exposing the insects. Termite collections can be kept for months in fruit jars with screen lids.

Termite gut pouches containing symbionts

To study the termite gut fauna, kill one by removing its head. Grab the thorax with fine forceps. Using another pair of forceps grab the tip of the abdomen and pull out the intestine. Look at the mid-portion of the gut and find the pouches that contain the cellulose digesting microbes. Place the intestine in a drop of .6 % saline solution. Add a cover glass and observe the pouches under low powers in transmitted light to see if you can learn where, in the pouch, the symbionts live. Use a bit of tissue paper to draw the saline solution from under the coverglass, compressing the gut for a better view. The squeeze may force some symbionts out of the intestine where they can be examined under higher magnification. Gut symbionts have very low tolerance for oxygen, so consider sealing the edges of the cover glass with warm petroleum jelly.

Microscope techniques for viewing of living protists

Prepared slides that have been stained to differentiate microscopic structures are best viewed using standard bright field illumination—the typical microscope lighting system. Using the microscope as you would for stained subjects, produces a ghost-like image of such low contrast that you can't see the internal cellular structures. There are several ways to increase contrast. The easiest method is to simply close down the iris diaphragm, increasing contrast although this is at the expense of theoretical resolution. Hovever, the loss of resolution is more than compensated for by actually being able to see cell structures that are otherwise virtually invisible.

Another way to increase contrast involves a trick known as “oblique lighting”. The idea is to light the subject more from one side than the other. If you have ever looked at the new moon with a telescope, you have noticed that along the terminator (the sunrise line) the mountains and craters stand out because the low angle of the sun creates shadows. The full moon looks quite flat. This is because, when full, the sun is shining directly down on the surface with no shadows to reveal the topography. To get the “now moon effect” you need to offset the light hitting the subject. Try cutting a half disc of black paper for the filter holder. Looking in the microscope with the condenser properly focused, split the field between bright and dark. Along the mid-line is a region where subjects will appear three dimensional due to oblique angle of light, and in high contrast due to the darker background. It's worth some experimentation in order to make observations using this extremely revealing form of microscope lighting.

bright field   aperture reduced   oblique   dark field

Dark field lighting is another excellent way to increase contrast in clear subjects. If your microscope has a condenser lens system, and a filter holder, creating dark field lighting is a snap. Just cut (or punch with a binder hole punch) a quarter inch circle of black paper. Glue it in the exact center of a piece of translucent plastic cut to fit the filter holder. Open the iris all the way and crank up the condenser until your subjects show up brightly lit against a black background. This technique is usually limited to the 4X and 10X objective lenses. High magnification dark field is best achieved with special dark field condensers. However, low magnification, dark field views of rotifers and other small invertebrates can be stunning. Not only does dark field lighting provide extreme contrast, but it allows you to see subjects in their natural color.

Observing protists in the field

But what if you don't have a microscope, or a stereo scope. Don't despair, you can study protist behavior with a hand lens, or better yet, an inexpensive hand held microscope called a DiscoveryScope. DiscoveryScope is a low power, single lens microscope with observation chambers for holding water samples. The chambers make acceptable homes for microorganisms, which

will live in them for days or week, allowing prolonged study of these little communities. A DiscoveryScope can be pointed at contrasting backgrounds, while the subjects are lit from behind. In this revealing dark field lighting even small protists such as Euglena can be recognized and their behavior studied.

Using DiscoveryScope

Stentor — as seen with DiscoveryScope